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Study of tRNA in maturing salmon (Oncorhynchus tschawytscha) testes Urquhart, Nadine Iris 1973

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I5l£<? A STUDY OF tRNA IN MATURING SALMON (Oncorhynchus tschawytscha) TESTES by NADINE IRIS URQUHART B.Sc, Un i v e r s i t y of B r i t i s h Columbia, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Biochemistry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1973 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis fo r scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of B/te 77«?y The University of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT During the sexual maturation of salmon testes, there i s a drastic decrease in the RNA content of testis tissue and there are marked changes in the type of proteins synthesized. For these reasons, maturing salmon testes seemed an excellent tissue in which to study developmental changes of tRNA popu-lations . Transfer RNA has been isolated from salmon testes at four distinct stages of sexual maturation. Determination of the amounts of tRNA in testes at these four stages of development has shown that during spermatogenesis the testis tRNA popula-tion decreases proportionally to that of total testis RNA. Evidence i s presented to suggest that the tRNA population of testis tissue i s specialized for the synthesis of basic nuclear proteins. Not only was the relative amount of arg-inine and lysine tRNA extracted from salmon testes found to be significantly greater than tRNA prepared from salmon li v e r but the relative amount of lysine and arginine tRNA in testis tissue at a specific stage of development seemed adapted to the specific type of basic nuclear protein synthesis occurring at that phase of maturation. These results, together with the results of others suggest that the levels of various tRNAs in c e l l s are altered in a manner consistent with the u t i l i z a -tion of various amino acids in protein synthesis. The tRNAAr^ isoacceptors of salmon testes were separated by chromatography on BD-cellulose and RPC-5 columns and their ribosome-binding responses compared in the presence of the six arginine codons which had been synthesized enzymatically. Evidence suggests that of the six tRNAAr^ isoacceptors sep-arated on RPC-5 arginyl-tRNA^. specific for CGA (C,U) and arginyl-tRNA^ specific for codon AGG preferentially increase during the phase of salmon testis maturation in which syn-thesis of basic nuclear proteins i s transformed from mainly histones to mainly protamines. Also, during this research, the effect of temperature, storage, NaCl concentration and ethanol on the extent of charging of salmon testis tRNA by salmon li v e r arginyl-tRNA synthetase was determined. The Km values of salmon l i v e r arginyl-tRNA synthetase for arginine and salmon testis tRNA were found to be 0.19 ]xM and 0.76 uM, respectively. i i i . TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS iii LIST OF TABLES vii LIST OF FIGURES ix ACKNOWLEDGEMENTS xi DEDICATION x i i ABBREVIATIONS USED x i i i INTRODUCTION 1 I. Process of Spermatogenesis 2 II. Process of Protein Synthesis and i t s Control . . 10 (a) Gene Reiteration or Amplification 16 (b) Transcriptional Control Mechanisms 17 (c) Post-transcriptional Control Mechanisms . . . 23 (d) Translational Control Mechanisms 28 III. Transfer RNA Adaptation for Specialized Protein Synthesis 42 MATERIALS AND METHODS 52 I. Chemicals and Instruments 52 (a) Chemicals 52 (b) Instruments 53 II. Biological Samples . . . . . 53 (a) Source of Salmon Tissues 53 (b) Histology of Salmon Testes 55 III. Isolation and Characterization of Salmon Testis Protein 55 iv. Page (a) A r g i n i n e I n c o r p o r a t i o n i n t o T e s t i s P r o t e i n 55 (b) F r a c t i o n a t i o n o f T e s t i s P r o t e i n 57 ( i ) A c i d E x t r a c t i o n o f T e s t i s P r o t e i n . . . . 57 ( i i ) B i o - G e l P-10 Chromatography 58 (c) R a d i o a c t i v i t y o f T e s t i s P r o t e i n F r a c t i o n s . . 59 (i) T o t a l A c i d I n s o l u b l e P r o t e i n 59 ( i i ) T o t a l H i s t o n e or T o t a l Protamine . . . . 60 (d) P o l y a c r y l a m i d e D i s c G e l E l e c t r o p h o r e s i s . . . 60 (e) P r o t e i n Content o f T e s t i s T i s s u e 61 (f) A r g i n i n e Content o f T e s t i s P r o t e i n 62 I I I . D e t e r m i n a t i o n o f DNA and RNA Content of Whole T i s s u e 62 IV. I s o l a t i o n of Salmon T e s t i s tRNA 63 (a) Whole C e l l tRNA 63 (b) Ribosomal tRNA 67 V. Assay of Amino A c i d A c c e p t o r A c t i v i t y of tRNA P r e p a r a t i o n s 68 (a) P r e p a r a t i o n o f Aminoacyl-tRNA Synthetase . . 68 (b) P u r i t y o f R a d i o a c t i v e Amino A c i d s 7 0 (c) Assay f o r Amino A c i d A c c e p t o r A c t i v i t y . . . 70 (d) Assay f o r True A r g i n i n e A c c e p t o r A c t i v i t y . . 72 V I . F r a c t i o n a t i o n o f Salmon T e s t i s A r g i n y l - t R N A . . . 74 (a) B D - C e l l u l o s e Chromatography 74 (b) Reversed-Phase Chromatography 74 V I I . Codon R e c o g n i t i o n o f Salmon T e s t i s A r g i n y l -tRNAs 76 Page (a) Preparation of Labeled Arginyl-tRNA . . . . . 76 (b) Concentration of Arginyl-tRNA Solutions . . . 78 (c) Preparation and Characterization of Arginine tRNA Codons 79 (i) Synthesis of Trinucleotides 79 (ii) Paper Chromatography and Electro-phoresis 81 ( i i i ) Characterization of Di and T r i -nucleotides 82 (d) Preparation of E. c o l i Ribosomes 83 (i) Growth of E. c o l i 83 (ii) Isolation of Ribosomes . . . . . . . . . 84 (e) Assay of Arginyl-tRNA Binding to Ribo-somes . 85 RESULTS AND DISCUSSION 87 I. Study of Spermatogenesis in 0. tschawytscha . . . 87 (a) Increase in Testis Size During Spermato-genesis 87 (b) Histology of Spermatogenesis . . . . . . . . 89 (c) Changes in Testis Proteins During Spermato-genesis 91 II. Transfer RNA Extraction • 103 III. Changes in Nucleic Acid During Spermatogenesis . 105 IV. Properties of Arginyl-tRNA Synthetase Prepara-tions I l l (a) Heat La b i l i t y of Salmon Liver Arginyl-tRNA Synthetase Preparation 112 (b) Stability of Salmon Liver Arginyl-tRNA Synthetase Preparations . . 114 vi. Page (c) K i n e t i c Properties of Salmon L i v e r Arginyl-tRNA Synthetase . . . . 114 V. VI. VII. (d) Assaying for True Arginyl-tRNA Formation . . 118 (e) The E f f e c t of Ethanol and Various NaCl Con-centrations on Arginyl-tRNA Formation . . . 123 (f) E f f e c t of Sephadex G-25 Chromatography on the A c t i v i t y of Salmon Liver and Salmon T e s t i s Arginyl-tRNA Synthetase Prepara-tions 125 Relationship of tRNA A r g and tRNA L y s to Basic Nuclear Protein Synthesis i n Salmon T e s t i s . . 127 Isoaccepting Forms of tRNA A r g i n Salmon T e s t i s at the Various Stages of Development 138 (a) BD-Cellulose Chromatography of tRNA A r g . . . 138 (i) Whole C e l l tRNA A r g 138 ( i i ) Ribosomal-bound tRNA A r g 145 (b) RPC-5 Chromatography of tRNA A r g 148 Codons Recognized by Salmon T e s t i s A r g i n y l -tRNAs 159 (a) Synthesis, P u r i f i c a t i o n , and Characterization of Codons by Arginyl-tRNAs 159 (b) Codon Recognition by Unfractionated A r g i n y l -tRNA 165 (c) Codon Recognition by an Arginyl-tRNA Frac t i o n from a BD-cellulose Column ... . . 169 (d) Codon Recognition by Arginyl-tNRA Fractions from a RPC-5 Column . . 17 0 CONCLUSIONS 181 BIBLIOGRAPHY 186 v i i . LIST OF TABLES Page Table 1. In v i t r o incorporation of [ 1 **C] -arginine into proteins of 0. tschawytscha testes 96 Table 2. Calculations of percentage of each type of pro-t e i n synthesized i n the four stages of salmon testes 97 Table 3. Percent arginine i n t o t a l protein of the various stages of 0. tschawytscha testes 100 Table 4. DNA content of tRNA Preparations . 104 Table 5. DNA and RNA values of O. tschawytscha testes and l i v e r during the various stages of sexual maturation 107 Table 6. RNA values of 0. tschawytscha testes and l i v e r during the various stages of sexual matura-t i o n 109 Table 7. S t a b i l i t y of salmon l i v e r arginyl-tRNA syn-thetase preparations 115 Table 8. Assay of true arginyl-tRNA formation 122 Table 9. The amino aci d acceptance a c t i v i t i e s of tRNA preparations from salmon l i v e r and various stages of salmon testes 130 Table 10. A comparison of the arginine and l y s i n e accep-tance a c t i v i t i e s of various t e s t i s tRNA prep-arations 135 Table 11. A comparison of the sizes of the two arginine acceptor peaks of t e s t i s tRNA preparations chromatographed on BD-cellulose columns . . . . 143 Table 12. Comparison of the arginine acceptor a c t i v i t y of unfractionated tRNA and BD-cellulose fractionated tRNA 146 Table 13. A comparison of the r e l a t i v e amounts and the actual amounts of s p e c i f i c arginyl-tRNAs i n stage 1 and stage 3 t e s t i s tRNA preparations . . 156 Table 14. Summary of the Rf values of the t r i n u c l e o t i d e . 163 v i i i . Page Table 15. Contamination of t r i n u c l e o t i d e s . 164 Table 16. Characterization of t r i n u c l e o t i d e s by RNase Tz digestion and estimation of molar r a t i o s . . . 166 Table 17. Characterization of t r i n u c l e o t i d e s by venom phosphodiesterase digestion and estimation of molar r a t i o s 167 Table 18. A c t i v i t y of unfractionated arginyl-tRNA from stage 3 salmon testes i n trinucleotide-stimu-l a t e d binding to E. c o l i ribosomes 168 Table 19. A c t i v i t y of arginyl-tRNA (tube 78 of BD-cellu-lose column f r a c t i o n a t i n g stage 3 t e s t i s tRNA) i n t rinucleotide-stimulated binding to E. c o l i ribosomes 171 Table 20. S p e c i f i c i t y of arginyl-tRNAs p u r i f i e d by RPC-5 chromatography i n trinucleotide-stimu-lated binding to E. c o l i ribosomes 176 ix. LIST OF FIGURES Page Figure 1. Change i n the t e s t i s weight of sexually maturing O. tschawytscha 88 Figure 2. Histology of developing 0. tschawytscha testes 90 Figure 3. Polyacrylamide gel electrophoresis of the t o t a l a c i d soluble proteins of 0. tschawytscha testes at various stages of spermatogenesis 92 Figure 4. Transformation from histone synthesis to protamine synthesis i n 0. tschawytscha testes , . 94 Figure 5. DEAE-cellulose chromatography of stage 3 t e s t i s tRNA 106 Figure 6. The e f f e c t of temperature on the extent of arginine acceptance of salmon t e s t i s tRNA i n 15 min when using a salmon l i v e r aminoacyl-tRNA synthetase preparation 113 Figure 7. The e f f e c t of arginine concentration upon the formation of arginyl-tRNA by a salmon l i v e r arginyl-tRNA synthetase preparation 116 Figure 8. The Lineweaver-Burk p l o t of a salmon l i v e r arginyl-tRNA synthetase preparation for determination of the Km values for arginine . . 117 Figure 9. The e f f e c t of salmon t e s t i s tRNA concentration upon the formation of arginyl-tRNA by a salmon l i v e r arginyl-tRNA preparation 119 Figure 10. The Lineweaver-Burk plot of a salmon l i v e r arginyl-tRNA synthetase preparation for deter-mination of the Km value for tRNA 120 Figure 11. The e f f e c t of ethanol and various NaCl con-centrations on arginyl-tRNA formation 124 Figure 12. The e f f e c t of Sephadex G-25 chromatography on arginine acceptor a c t i v i t y of aminoacyl-tRNA synthetase preparations . 126 X. Page Figure 13. Relative amounts of the various tRNAs during maturation of salmon testes 136 Figure 14. E l u t i o n p r o f i l e s of stage 4 t e s t i s tRNA prep-arations on BD-cellulose columns i n the presence and absence of dimethylformamide . . . 139 Figure 15. A comparison of the arginine acceptor p r o f i l e s of tRNA from various stages of testes chrom-atographed on BD-cellulose columns . . . . . . 142 Figure 16. A comparison of the arginine acceptor p r o f i l e s of bulk tRNA and ribosomal tRNA from stage 2 testes chromatographed on BD-cellulose columns 149 Figure 17. RPC-5 p r o f i l e of stage 3 salmon t e s t i s a r g i n y l -tRNA 151 Figure 18. RPC-5 p r o f i l e s of arginyl-tRNAs from stage 1 and stage 3 testes 153 Figure 19. RPC-5 p r o f i l e s of arginyl-tRNAs from stage 3 testes 155 Figure 20. Arginyl-tRNA p r o f i l e s on a RPC-5 column i n -d i c a t i n g the exact amount of s p e c i f i c a r g i n y l -tRNAs i n stage 1 and stage 3 t e s t i s tRNA preparations 158 Figure 21. P u r i f i c a t i o n of the t r i n u c l e o t i d e CpGpG by DEAE-cellulose chromatography 161 Figure 22. Uncharged salmon t e s t i s tRNA chromatographed on a RPC-5 column 173 Figure 23. A large scale RPC-5 chromatographic separation of arginyl-tRNAs from stage 3 salmon testes . . 175 ACKNOWLEDGEMENTS I wish to thank my research supervisor Dr. Michael Smith for his guidance and encouragement during the course of this work. His sincere interest in students and his eager-ness for s c i e n t i f i c knowledge w i l l be remembered. I am also indebted to Drs. Caroline A s t e l l , Shirley Gillam, Ian Gillam, Bradley White, and Gordon Tener for valuable discussions during the course of this work. A special thanks to my husband David for the preparation of a l l illustrations in this thesis. I wish to express my appreciation for the helpful co-operation of Mr. C.F.A. Culling, Department of Pathology, University of Brit i s h Columbia in preparation of testis samples for histological examination. I also wish to thank Mr. Vivian Wylie, the Canadian Department of Fisheries, Mr. Steve F a l l e r t of the Green River Hatchery, Washington State Department of Fisheries, and the commercial Fraser River fishermen, Ernie Dragsund and Leonard De Gans for assistance in collecting salmon testes, livers and milt. I was the recipient of a H.R. MacMillan Family Scholar-ship (1967-1968) and a Medical Research Council of Canada Studentship (1968-1972). DEDICATION t o husband David and my parents ABBREVIATIONS USED The abbreviations summarized below are those suggested by the IUPAC-IUB Combined Commission on Biochemical Nomen-clatu r e [Revised Tentative Rules, 1965 (1) and Recommendations, 1970 (2)]. Recommendations (1970) (2) replace Section 5 of the Revised Tentative Rules (1965) (1). A, C, G, U:- the ribonucleotides of the four bases; adenine, cytosine, guanine, and u r a c i l . pA (or AMP), pC, pG, pU:- the 5'-ribonucleoside monophosphate Ap, Cp, Gp:- the 3 *-ribonucleoside monophosphate ADP, CDP, GDP, UDP:- the 5'-ribonucleoside diphosphates. ATP, GTP:- The 5*-ribonucleoside triphosphates p:- phosphate P i : - " inorganic orthophosphate PPi:- inorganic pyrophosphate poly(G):- polyriboguanylic a c i d . Poly(A) and poly(U) are analogous struc-tures. poly (A,G) :- polyribonucleotide with random sequences of adenylate and guany-l a t e residues. Poly(U,C) and poly(U,G) are analogous structures. DNA: - deoxyribonucleic acid RNA: - rib o n u c l e i c acid xiv. rRNA:-mRNA:-tRNA:-tRNA A r g:-Arg-tRNA or Arg-tRNA tRNA^ 5: -fMet-tRNA Met Met-tRNA Met f* Ala, Arg, Asp, Asn, Glu, Gly, Leu, Lys, Met, Phe, Ser, Tyr Tr i s : -EDTA:-CM-cellulose:-DEAE-cellulose:-ribosomal ribonucleic acid messenger ribonucleic acid transfer ribonucleic acid nonacylated arginine tRNA aminoacylated arginyl-tRNA one of the isoaccepting species of tRNA A r g prokaryotic i n i t i a t o r tRNA, formy1-methionyl-tRNA eukaryotic i n i t i a t o r tRNA, methionyl-tRNA f* M e t amino acid residues, alanine, arginine, aspartic acid, aspara-gine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, and tyrosine, respectively, tris(hydroxymethyl)aminomethane ethylenediaminetetraacetate 0-(carboxymethyl) cellulose 0-(diethylaminoethyl) cellulose ZTCA: -Other abbreviations and definitions are: trichloroacetic acid XV . TCA-tungstate:-TMKS:-BD-cellulose: A26o nm:-A.U.:-A 2 6 0 unit:-Rf :-w/v: -v/v:-RNase; e :-C-G:-a precipitant for protein des-cribed by Gardner et a l . (3) con-taining 5 % (w/v) TCA and 0.25 % (w/v) sodium tungstate, f i n a l pH, 2.0 an isotonic medium containing Tris HC1 50 mM (pH 7.6), magnesium acetate 5 mM, potassium chloride 25 mM, and sucrose 0.25 M. benzoylated DEAE-cellulose absorbance at 260 nm absorbance unit the amount of material giving an absorbance of 1.0 in 1.0 ml of solution at neutral pH in a 1 cm light path at 260 nm. mobility relative to the solvent front weight per volume volume per volume ribonuclease the molar extinction coefficient, equal to the absorbance of a 1 molar solution in a 1 cm light path. guanine-cytosine base pair xvi. G-G:- guanine-guanine base pair dRNA:- DNA-like RNA HnRNA:- heterogeneous RNA UC(U,C,A)-specific tRNA:- tRNA responding to UCU, UCC, UCA codons in ribosome binding assay. A system of nomenclature for the protein synthetic factors that was recently agreed upon by workers in the f i e l d (4) has been followed in this thesis. The nomenclature system for histones of Rasmussen, Murray, and Luck (5) was followed in this thesis. Classification of salmonids: Genus Oncorhynchus (Pacific salmon) Species tschawytscha (chinook) keta (chum) nerka (sockeye) Genus Salmo (Atlantic salmon) Species gairdnerii (rainbow trout) 1. INTRODUCTION This thesis describes an investigation of the changes in the tRNA population of salmon testes during spermatogenesis. The f i r s t part of the introduction describes the s u i t a b i l i t y of the salmon testis system for such a study noting especially the drastic decrease in RNA and the transformation of basic nuclear proteins from histones to protamines that occurs during the sexual maturation of salmon testes. The second part of the introduction discusses the process of protein synthesis and i t s control mechanisms. These mechanisms include the control of transcription, maturation, transport, degradation and trans-lation of cellular RNA. In the third and last part, studies on the tRNA of differentiated c e l l s are described suggesting the adaptation of the tRNA pool of a c e l l to the amino acid composition of the proteins synthesized in the c e l l s . 2. I. Process of Spermatogenesis The general scheme of spermatogenesis i s represented by the following diagram ( 6 ) . meiosis mitosis 1st meiotic SPERMATOGONIA -> PRIMARY -SPERMATOCYTE d i v i s i o n ^ SECONDARY SPERMATOCYTE (2n) I 2nd | meiotic l, d i v i s i o n spermiogenesis SPERMATOZOA SPERMATIDS V (n) The process involves a p r o l i f e r a t i o n of the spermatogonia (germ c e l l s ) through repeated mitoti c d i v i s i o n s and growth to form primary spermatocytes; each primary spermatocyte then undergoes meiosis which leads via . two d i p l o i d secondary spermatocytes to the formation of four haploid spermatids. These spermatids then metamorphose into motile and p o t e n t i a l l y functional gametes — spermatozoa — by the process of spermiogenesis. The process of spermiogenesis consists of a series of complex changes i n c e l l morphology, which include the conservation of nuclear, mitochon-d r i a l and axonemal components and the elimination of excess cytoplasm and nucleoplasm. For example, during spermiogenesis i n f i s h , the Golgi apparatus develops into the acrosome, the nucleus condenses and forms with the acrosome the head of the sperm c e l l , and the mitochondria and c e n t r i o l e s both p a r t i c i p a t e i n constructing the t a i l ( 7 ) . The process of spermatogenesis i n salmonids has been studied by various investigators (8-11) and ind i c a t i o n s are that i t 3. follows the general scheme as diagramed above. Unlike the mammals, however, where spermatozoa are usually formed contin-uously; in salmonids, large numbers of spermatozoa are accumu-lated in the testes only in anticipation of spawning. Salmonids produce these spermatozoa by means of a cystic type of germ c e l l production, characteristically displayed by a l l anamniotic species (12) . In these species, germ cel l s proliferate in co-ordinated clusters enclosed in membranous cyst walls. Within any one cyst the germ c e l l s are a l l at the same stage of develop-ment and maturing at the same rate. Spermatogenesis i s an example of hormonally controlled cellular differentiation. A l l investigators agree that the gonadotrophic hormone(s) of the adenohypophysis of the pituitary are required for testis maturation of teleosts (13). In the absence of a pituitary, spermatogenesis i s blocked at the spermatogonia-spermatocyte stage and steriodogenesis does not occur in the testicular endocrine tissue. Spermatids can under-go spermiogenesis in absence of a pituitary but spermiation i s not observed (14,15). The testes of immature salmon respond readily to treatment with extracts of pituitary glands obtained from spawning salmon (16,17). Injection of crude pituitary extracts (17) or implantation of par t i a l l y purified prep-arations (16) into Salmo gairdnerii produced complete maturation of testes with shedding of motile spermatozoa in two months' time. The major external stimulus of the sexual maturation in salmonids i s the photoperiod which acts through the hypothalmus 4 . controlling the gonadotropin secretion (18). The characteristic morphogenetic changes which occur during sperm formation are accompanied by profound chemical alterations of the nuclear material. During the latter stages of salmonoid spermatogenesis, the typically somatic-type histones, arginine-lysine rich structural proteins of the chromatin, are replaced by protamines, a class of sperm specific, small proteins (average molecular weight 5,000) very rich in arginine (19). A l l histones contain substantial amounts of lysine while the arginine content varies from low in histone I (0.9%) to high in histone III (11.5%) and IV (12.5%) (20)* In contrast, protamines are extremely rich in arginine, for of the 33 amino acid residues of Oncorhynchus  tschawytscha protamines, 22 are arginine (19). There are only six other amino acids present — serine, proline, glycine, alanine, valine, isoleucine — and these are a l l neutral; there are no aromatic, acidic or sulphur containing amino acids. Since protamines appear during the transformation of the male repro-ductive c e l l s into metabolically inert spermatozoa, the much higher content of arginine in protamines as compared with somatic histones can be interpreted as the basis for the mechanism by which the DNA in spermatozoa i s kept tightly packed for i t s delivery during f e r t i l i z a t i o n (21). This replacement of histones by protamine was observed by Miescher (22) and others (23-25). Alfert (8) using cytochemical techniques, showed that the protamines appeared in middle stage Arginine composition of histone fractions of trout testis chromatin. 5. spermatids of 0. tschawytscha and associated with their appear-ance was the loss of histone staining properties. By separating trout testis c e l l s on serum albumin gradients and analyzing their basic nuclear proteins on starch gel, Louie and Dixon (26) characterized the acid soluble nuclear proteins present in and synthesized by each c e l l type. Histones were the only basic nuclear protein present in and synthesized by the spermatogonia and spermatocytes. Histones were shown to gradually disappear from the spermatid c e l l s during their maturation. Coincident with histone disappearance was the rapid synthesis of protamine by maturing spermatids. Thus, at the completion of spermiogenesis, mature sperm contained only protamine. Despite i t s small size and unusual composition, protamine was found to be synthesized by the usual mechanism of protein synthesis involving mRNA, ribosomes and tRNA (25). Thus, protamine synthesis occurs in the cytoplasm of testis c e l l s (27) and i s very sensitive to inhibition by cycloheximide (25) . Methionine is the i n i t i a t i n g amino acid in protamine synthesis (28); the same amino acid known to be the universal i n i t i a t o r of protein synthesis. Ling and Dixon (29) have implicated a special class of cytoplasmic polysomes, the diribosomes (disomes), sedimenting at 120 S as the species of polysome synthesizing protamine. Protamines after synthesis are quickly transported into the nucleus where they replace the histones on the DNA (25). Since actinomycin D p a r t i a l l y inhibited protamine synthesis during an 6. e a r l y stage of development but d i d not i n h i b i t protamine s y n t h e s i s d u r i n g the a c t i v e protamine s y n t h e s i z i n g stage, L i n g and Dixon (29) have suggested t h a t the mRNA f o r protamine i s s y n t h e s i z e d immediately b e f o r e the main stage o f protamine s y n t h e s i s takes p l a c e and i s m e t a b o l i c a l l y s t a b l e . In a d d i t i o n to the g e n e r a l replacement of h i s t o n e s by protamines d u r i n g spermatogenesis, a change i n the h i s t o n e com-p o s i t i o n o f chromatin has been observed d u r i n g t r o u t t e s t i s m a t u r a t i o n (30). The types o f h i s t o n e s i n t r o u t t e s t e s a t e a r l y stages o f spermatogenesis was found to be q u a n t i t a t i v e l y s i m i l a r t o those i n t r o u t l i v e r . However, as the t r o u t t e s t e s matured, the r e l a t i v e c o n c e n t r a t i o n o f l y s i n e - r i c h h i s t o n e I i n c r e a s e d , w h i l e the r e l a t i v e c o n c e n t r a t i o n o f a r g i n i n e - r i c h h i s t o n e IV decreased. Hi s t o n e T, a newly c h a r a c t e r i z e d h i s t o n e e s p e c i a l l y r i c h i n l y s i n e and a l a n i n e , a l s o i n c r e a s e d i n r e l a t i v e amount j u s t b e f o r e the protamine replacement o f h i s t o n e o c c u r r e d i n t r o u t t e s t e s (31). The RNA con t e n t of salmonoid t e s t e s decreases as the t i s s u e matures. Creelman and Tomlinson (32) found t h a t the c o n c e n t r a t i o n of RNA phosphorus i n Oncorhynchus nerka t e s t e s , t w o - t h i r d s mature on a weight b a s i s , was 43.5 mg per 100 g; whereas i n f u l l y mature t e s t e s i t was 17.4 mg per 100 g of t i s s u e . The concen-t r a t i o n o f RNA phosphorus i n the l i v e r o f the same f i s h was c o n s t a n t a t approximately 8 0 mg per 100 g of t i s s u e d u r i n g the same p e r i o d . T h i s i n d i c a t e d t h a t the RNA c o n t e n t o f the l i v e r , u n l i k e t h a t o f the t e s t e s , i s independent of the sex u a l matura-t i o n o f the f i s h . 7. It i s characteristic of spermatogenesis that RNA disappears from maturing spermatids. For example, Ando and Hashimoto's studies (29) on testes of maturing Salmo gairdnerii showed that the concentration of RNA phosphorus in nuclei decreased from a value of 0.24% of the dry weight of the nucleus at an inter-mediate stage of development (probably corresponding to the late spermatogonia to primary spermatocyte stage) to 0.05% of the dry weight of the nucleus at the spermatid stage when protamine i s produced. The nuclei of mature salmonoid sperm are essentially devoid of RNA (24,33-35). Louie and Dixon (26), who determined the RNA content of Salmo gairdnerii testis c e l l s , also found a progressive decrease in RNA as germ c e l l s mature to spermatozoa. The RNA content decreased from 1.9 picograms/cell in the spermatogonia/ spermatocyte c e l l s to 0.23 picograms/cell in spermatids actively synthesizing protamine to a value of zero in mature sperm. Consistent with a l l these observations, Ling and Dixon (29) found a progressive loss of ribosomes from the cytoplasm of developing rainbow trout sperm c e l l s . The content of ribo-somes per gram of testes decreased during development from a value of 1.95 mg for preprotamine testis, to 0.71 mg for testes actively synthesizing protamine, to f i n a l l y 0.03 mg for mature testes. This removal of RNA from developing sperm cel l s may be a specific and well regulated process because the decrease in RNA associated with Oncorhynchus nerka testis maturation i s 8. accompanied by a change in base composition; the RNA in mature tissue being richer in guanosine (32). A progressive decrease in RNA synthesis was noticed in maturing testis c e l l s of mammals (36) and insects (37) . For instance, Monesi (38) from studies on [ 3H]-uridine incorporation into the RNA of mouse testes noted that RNA synthesis was f a i r l y rapid in spermatogonia and primary spermatocytes, but decreased to a low value in secondary spermatocytes and early spermatids. Ke found no RNA synthesis in maturing spermatids. Also, though RNA synthesis was found to occur in early post-meiotic spermatids of the ram (36) and the grasshopper (37), in both cases, as soon as nuclear condensation and elongation has begun, no further synthesis of RNA occurred in the spermatid. By following the evolution of labeled RNA in mouse testes, Monesi (38-48) found that the RNA's synthesized during meiosis and early spermiogenesis are completely lost from the nucleus by breakdown and by transfer to the cytoplasm. The resulting intermediate and late spermatids are characterized by an elongating nucleus, devoid of RNA and a cytoplasm mass dis-placed at one end of the head containing f a i r amounts of RNA that were synthesized at least a week earlier during meiotic prophase. In very late spermiogenesis, this labeled RNA of meiotic orgin i s concentrated in larger and larger granules and then forms the so called "residual bodies" of Regaud (41), which are then extruded from the c e l l shortly before the release of the mature spermatids in the lumin. Thus the spermatids of mammals, insects and salmonids cease RNA synthesis and e f f i c i e n t l y 9. eliminate RNA as maturation occurs. In contrast to the other studies (36-38), Abraham and Bhargava (4 2) found that mature spermatozoa (bovine) synthesize RNA at a significant rate in vi t r o . The recent study by Premkumar and Bhargava (43) indicates that the RNA synthesis by mature bovine spermatozoa of 4 S, 16 S and 23 S RNA's, resembl-ing bacterial tRNA and rRNA in various fractionation systems, is completely mitochondrial in origin. They, like other researchers, found that the nuclear DNA was not actively trans-cribed in mature spermatozoa. 10. II. Process of Protein Synthesis and i t s Control Mechanisms It i s generally considered that the phenotype of a d i f f e r -entiated c e l l i s determined by i t s particular complement of proteins. The diversity of both form and function displayed by differentiated c e l l s i s the result of cyto-differentiation. This i s a controlled process which involves the selective synthesis of specialized proteins in different c e l l s at different times. Consequently the understanding of the mechanism of protein synthesis and i t s regulation has become a central problem in modern biology. During the last ten years, molecular biologists have largely elucidated the mechanism of protein synthesis. Only a brief summary of the known steps in protein synthesis w i l l be given here as detailed accounts have been given recently (44,31) . In a l l organisms, with the exception of RNA phages and RNA viruses, DNA i s the storehouse of genetic information. It is the molecule that determines the nucleotide sequence of a l l RNA's and the amino acid sequence in a l l proteins of an organism. The nucleotide sequence of DNA i s f i r s t transcribed by DNA-dependent RNA polymerase, forming a complementary RNA (mRNA) which carries genetic information to the cytoplasm of the c e l l , the site of protein synthesis. Here, specific nucleotide sequences are translated into amino acids of particular proteins. 11. The f i r s t step in protein synthesis, discovered by Hoagland (45), involves the formation of an enzyme-bound aminoacyl-adenylate complex (reaction 1) in which the amino acid carboxyl group forms an anhydride with the phosphate of AMP. This i s followed by the transfer of the aminoacyl moiety to a specific tRNA (reaction 2); i.e., the carboxyl group of the amino acid is linked by an ester bond to the hydroxyl group of the ribose of the terminal adenylic acid residue of tRNA. 1. Amino acid + ATP + enzyme K > aminoacyl-AMP-enzyme + PPi 2. Aminoacyl-AMP-enzyme + tRNA^— > aminoacyl-tRNA + AMP + enzym Both steps are catalyzed by a single enzyme, an aminoacyl-tRNA synthetase, which i s essentially specific for the amino acid and tRNA involved. The tRNA molecules transport the amino acids to the mRNA template at the ribosomes, the site of translation. Each tRNA contains an anticodon region in the form of a tr i p l e t of bases specific for the amino acid which i t carries and by i t s interaction with a t r i p l e t codon in mRNA translates the genetic message into a polypeptide sequence. The i n i t i a t i o n mechanism in protein synthesis ensures that each ribosome associates with a mRNA molecule at the correct codon (initiator codon), preventing nonsense proteins from being synthesized. Because the present evidence on eukaryotic protein i n i t i a t i o n is s t i l l fragmentary, the better known mechanism of bacterial i n i t i a t i o n w i l l be presented. In prokaryotes i n i t i a t i o n of protein synthesis is thought to require three i n i t i a t i o n factors (IF-1, IF-2, IF-3) and the formation of a 30 S subunit*mRNA • fmet-tRNA complex that i s converted to a 7 0 S i n i t i a t i o n complex by the addition of a 50 S subunit (44). Initiation factor IF-1 i s apparently bound to the 30 S subunit in the 70 S particle, stimulating the rate of dissociation of the 70 S ribosome (46) . Factor IF-3 then binds to the free 30 S subunit (47) and acts as an anti-association factor (48) by preventing i t from combining with a 50 S particle. Subsequent attachment of the complex [IF-2'fmet-tRNA] to the [30 S*IF-1*IF-3] complex strengthens the binding of IF-3, shifting the equilibrium greatly towards the formation of [3 0 S•IF-1•IF-3•IF-2•fmet-tRNA] complex that attaches to the AUG i n i t i a t o r codon of mRNA (46). The i n i t i a t i o n factors now leave the 30 S subunit in the form of an [IF-2*IF-3] complex (4 9) and the 50 S subunit joins the 30 S complex in a reaction that requires GTP. It should be noted that this i n i t i a t i o n model recently proposed by Noll (46) , in contrast to previous views (44,31), stipulates that fmet-tRNA binding precedes, rather than follows, the binding of messenger RNA. Experiments exploring the i n i t i a t i o n mechanism in eukaryotes have demonstrated that the process of i n i t i a t i o n in eukaryotes i s essentially similar to that of prokaryotes. Just as in bacteria, cyclic dissociation of ribosomes into subunits, l i k e l y occurs between the rounds of polypeptide chain i n i t i a t i o n in higher organisms (50). Also, as in prokaryotes, three i n -i t i a t i o n factors (IF-M1, IF-M2, IF-M3) have been extracted with 0.5 M KC1 from crude eukaryotic ribosomes (51,52). Experiments (53,28) have also indicated that in protein syn-thesis, methionine is the universal eukaryotic, as well as Met prokaryotic i n i t i a t i n g amino acid. F-met-tRNAf appears to i n i t i a t e synthesis of a l l bacterial proteins (54-57) and probably a l l proteins in mitochondria (58,59) and chloroplasts (60). However, in the cytoplasm of eukaryotes the specific Met i n i t i a t o r tRNA, met-tRNAf# i s not formylated (61-63). In contrast to the i n i t i a t i o n step, relatively complete studies with eukaryotic elongation factors (EF-1 and EF-2) have defined a series of partial reactions involved in protein elongation. EF-1, the f i r s t factor involved in the elongation mechanism has been isolated in two forms from calf brain (64). EF-lA, a multimeric molecule with a molecular weight in excess of 150,000 daltons apparently interacts with GTP to form a [EF-1A'GTP] complex which in turn forms a ternary complex with aminoacyl-tRNA. During the formation of this ternary complex, the elongation factor dissociates to yield the lower molecular weight (60,000 - 80,000) species EF-1B. As the amino-acyl-tRNA binds to the acceptor site of the ribosome, where i t recognizes i t s appropriate codon, the [aminoacyl-tRNA'EF-1B-GTP] complex dissociates and the GTP is hydrolyzed to GDP. The liberated [EF-1B-GDP], by reacting with GTP, seems then to dimerize and be reconverted to [EF-lA *GTP]. At the next step in elongation, peptidyl transferase, an enzyme which is an integral part of the 60 S ribosomal subunit (65,66), catalyzes the formation of the peptide bond between the a-amino group of the incoming aminoacyl-tRNA and the carboxyl-terminal group of the neighbouring i n i t i a t o r or peptidyl-tRNA. In the presence of elongation factor EF-2 and GTP the newly formed peptidyl-tRNA and mRNA are translocated from the acceptor site to the peptidyl site of the ribosome. This liberates the acceptor site of the ribosome allowing the acceptance of another aminoacyl-tRNA and exposing a new codon in the mRNA. Simultaneously with peptidyl-tRNA translocation, the deacylated tRNA is released from the peptidyl site of the ribosome and GTP i s hydrolyzed (44). L i t t l e i s known about how the EF-2 factor interacts with ribosomes, mRNA and peptidyl-tRNA to cause the trans-location reaction. Because the present evidence on eukaryotic peptide term-ination i s fragmentary, the better known mechamism of bacterial termination reviewed in (44) w i l l be discussed below. Peptide termination allows the completed protein to be released from the ribosomal•peptidyl-tRNA complex when i t reaches one of the terminator codons (UAA, UAG, or UGA) on a mRNA. Experimental evidence indicates that a release factor forms a R-factor•term-inator codon-70 S ribosome intermediate during terminator codon recognition. Three release factors are associated with chain termination in prokaryotes. RF-1 and RF-2 are codon specific; they bind to ribosomes in the presence of UAG and UAA, and UAA and UGA, respectively. A third release factor, RF-3 stimulates the binding of RF-1 and RF-2 to the appropriate termination codon. RF-1 and RF-2 when bound to ribosomes appear to convert the peptidyl transferase into a hydrolase, causing the peptidyl moiety of the peptidyl-tRNA to be trans-ferred to water rather than an aminoacyl-tRNA. Now, a factor called TR removes the discharged tRNA from the peptidyl site of the ribosome (67) . The two heat stable factors (EF-G, the elongation factor and RR) (68) and GTP appear to be required for the release of the ribosomes from mRNA. Eukaryotic peptide termination appears fundamentally similar to that in bacteria. However, in mammalian c e l l s , a single protein fraction, having a specific requirement for GTP, rec-ognizes a l l three termination codons. Although, the mechanism of protein synthesis in eukaryotes is now largely understood, the regulation of protein synthesis in eukaryotes s t i l l remains one of the major unresolved problems of biology. Experimental evidence indicates that in eukaryotes there are several levels at which mechanisms controlling the flow of genetic information can operate. The transfer of information can be controlled at the levels of gene reiteration, gene replication, gene transcription, RNA maturation, mRNA translation and protein activation. Transcriptional mechanisms controlling the expression of genetic information operate at the DNA level and determine the amount of tRNA, rRNA and mRNA potentially available to the eukaryotic c e l l . Post-trans-criptional mechanisms control the actual amount of specific RNA species available to the c e l l through the regulation of matura-tion, transport, and degradation of the transcribed RNAs. Translation mechanisms operate at the polysome level and control, the translation of mRNA. Post-translational mechanisms (e.g. cleavage of i n i t i a l translation product to an active protein) operate at the level of the protein molecule and control the actual amount of active protein in a c e l l . In. fact, a l l types of regulation are l i k e l y important for the expression of a phenotypic characteristic in a highly specialized c e l l . Those mechanisms that l i k e l y control protein synthesis w i l l be detailed in the following section. (a) Gene Reiteration or Amplification In higher organisms a single c e l l type i s often called on to produce a large amount of a single protein. Two ways in which this might be accomplished are: (i) the presence, in the DNA of a l l c e l l s , of multiple copies of the relevant genes (reiteration) and (ii) the specific replication of the required gene in the c e l l which synthesizes the protein (amplification). A precedent for both is established because the genome of every higher organism examined contains multiple ribosomal genes, and in both amphibians and insects the ribosomal genes are further amplified by a specific replication during oogenesis (69,7 0). However, apart from histone genes which appear to be highly reiterated (71), most genes specifying proteins in higher organisms are not reiterated unless to a very small extent (72-74). Molecular hybridization experiments between duck DNA and duck hemoglobin mRNA (72) or between cDNA (partial copy of 9 S RNA from mouse reticulocytes obtained using reverse transcriptase) and mouse embryo DNA (73) showed that there i s l i t t l e or no reiteration of hemoglobin genes in the duck or mouse. Similarly, molecular hybridization experiments between purified fibroin mRNA and Bombyx mori DNA showed only one to three fibroin genes per haploid complement (74) , indicating again that there i s l i t t l e or no reiteration of genes speci-fying specialized proteins. Furthermore, the relative abundance of DNA nucleotide sequences complementary to fibroin mRNA was the same in DNA from the animal's carcass, the middle s i l k gland or the posterior s i l k gland where fibroin i s synthesized in vivo (74) . Likewise, no specific gene amplification was detected in immature duck red blood c e l l s which were actively synthesizing hemoglobin mRNA (72). (b) Transcriptional Control Mechanisms For about 12 years, i t has been known that in bacteria the rate of synthesis of a specific protein i s regulated mainly by controlling the rate of synthesis of the mRNA coding for the protein (75) . However, since the average propagation rate of a l l RNA chains in E. c o l i i s the same, being 15-26 nucleotides per second at 29° (76), differences in total synthesis of RNA fractions in bacteria reflect differences in rates of i n i t i a t i o n . Bacteria control the i n i t i a t i o n of gene transcription by using repressor and activator molecules. One such repressor i s the lac repressor protein of E. c o l i . This repressor binds to lac operator DNA (77-79) preventing RNA polymerase (which binds at the preceding and adjacent site on the DNA) from pro-ceeding along the lac operon to synthesize mRNA (80) . The genes of the lac operon are transcribed only when the inducer removes the repressor from the lac operator DNA (81). Another auxiliary transcription factor i s adenosine-3',51 cyclic phosphate receptor protein (82) , which in the presence of adenosine-3 1,5 1 cyclic phosphate helps the RNA polymerase i n i t i a t e transcription at promoter sites of genes subject to catabolite repression for which i t s a f f i n i t y i s normally low. On the other hand, during bacteriophage development and bacterial sporulation — two cases of programmed transcription — sequen-t i a l changes in gene transcription are achieved by altering the transcription machinery, i.e., the RNA polymerase. E. c o l i RNA . polymerase has been isolated in two forms (83,84); as a cata-l y t i c unit (core enzyme) whose subunit structure i s $',$, 2 a , possessing a limited capacity to transcribe certain restrictive templates (e.g. T4 DNA) in vitro and as a holoenzyme ( 8 ' 8 a 2 e ) con-taining the G subunit. The <Tsubunit or sigma factor associates transiently with core polymerase and confers on i t the a b i l i t y to i n i t i a t e transcription of specific genes (83,85). Changes in the transcription machinery include the functional replace-ment of one sigma factor by another conferring a different i n i t i a t i o n specificity on the core polymerase. During phage T4 infection the E. c o l i sigma factor directs the synthesis of immediate early phage RNA and i s then altered or inactivated (86). Then an e a r l y phage T4 sigma f a c t o r appears which d i r e c t s the s y n t h e s i s of d e l a y e d e a r l y RNA, t o g e t h e r w i t h a s u b c l a s s of immediate e a r l y RNA (87) . Changes a l s o i n c l u d e m o d i f i c a t i o n of the RNA polymerase core enzyme. For example, begi n n i n g 2 min a f t e r phage T4 i n f e c t i o n , the a s u b u n i t of E. c o l i RNA polymerase i s a d e n y l a t e d (88) . A l s o d u r i n g B. s u b t i l i s s p o r u l a t i o n , the 3 s u b u n i t of i t s polymerase i s m o d i f i e d (89) . Changes i n the t r a n s c r i p t i o n machinery a l s o i n c l u d e the s y n t h e s i s of a new polymerase. E a r l y i n phage T7 i n f e c t i o n a new polymerase coded f o r by gene 1 of T7 i s syn-t h e s i z e d . T h i s T7 polymerase i s r e q u i r e d f o r the t r a n s c r i p t i o n of the remainder of the T7 genome (90,91). The e u k a r y o t i c chromosome, u n l i k e the b a c t e r i a l chromosome, c o n t a i n s a t l e a s t as much permanently a s s o c i a t e d p r o t e i n as DNA. H i s t o n e s and non-histones are the two c l a s s e s of p r o t e i n s a s s o c i a t e d w i t h the e u k a r y o t i c DNA. The f u n c t i o n o f the h i s t o n e s i s unknown, but t h e i r a s s o c i a t i o n w i t h DNA l e a d s to the form-a t i o n of a compact, p o s s i b l y s u p e r c o i l e d , s t r u c t u r e (92) which i s r e l a t i v e l y i n a c t i v e i n RNA s y n t h e s i s (93) . Paul (94) b e l i e v e s t h a t to f a c i l i t a t e t r a n s c r i p t i o n , the n e u t r a l i z a t i o n of t h i s e f f e c t of h i s t o n e s i s r e q u i r e d . Much evidence suggests t h a t non-histone chromatin p r o t e i n s perform t h i s f u n c t i o n . A c t i v e t i s s u e s c o n t a i n more non-histone chromatin p r o t e i n s than i n -a c t i v e ones (95). Non-histone chromatin p r o t e i n s r e s t o r e h i s t o n e -i n h i b i t e d DNA-dependent RNA s y n t h e s i s (96) . In f a c t , non-h i s t o n e chromosomal p r o t e i n s i n v i t r o are capable of d i f f e r -entially augmenting transcription from DNA sequences of con-densed chromatin (97) . It has also been shown that non-histone chromatin proteins are tissue specific (98) . Further-more, the rate of synthesis of non-histone chromatin proteins in l i v e r c e l l s i s correlated with activity and more specifically with the extent to which li v e r c e l l s are engaged in inform-ational RNA synthesis (99,100). Thus, Paul has proposed in a recent model (94) that non-histone proteins may bind at address l o c i (repetitive sequences in eukaryotic DNA) of the eukaryotic genome causing localized reductions in supercoiling allowing RNA polymerase molecules to bind to promoter sites. Additional regulatory substances may help i n i t i a t e or inhibit the RNA polymerase progression to the i n i t i a t o r site on the genome. Steroids may be substances that help control gene trans-cription in eukaryotes because one of their fundamental actions i s to induce increases in specific mRNAs which in turn results in the synthesis of specific proteins. For example, proges-terone induces accumulation of avidin mRNA in the oviduct target c e l l s (101) while estrogen induces accumulation of ovalbumin mRNA in oviduct target c e l l s (102). Furthermore, in accord with Paul's model (94), the progesterone-receptor complex of oviduct c e l l s becomes associated with oviduct chromatin (103), binding to the non-histone proteins associated with the DNA (104). Further evidence that regulatory substances may control gene transcription in eukaryotes came from studies on somatic c e l l hybridization (105,106). Davidson found that when d i f f e r -entiated c e l l s which synthesize a tissue-specific protein (dopa-oxidase, growth hormone, or S100) are hybridized with undifferentiated c e l l s which do not synthesize the protein, the activity of the protein i s absent or reduced by at least 90 percent in the hybrids. Therefore, the results of hybrid-izing differentiated and undifferentiated c e l l s suggest that the absence of proteins characteristic of a given c e l l ' s differen-tiated function may be due to the production, by a genome in the undifferentiated c e l l , of a diffusible regulator substances which specifically represses the expression of the relevant genes. Three distinct species of RNA polymerase have been detected in the nuclei of a variety of eukaryotic c e l l s (107-110). Polymerase I, located in the nucleolus and a-amanitine resis-tant i s probably responsible for ribosomal RNA synthesis (108). Polymerase II, a-amanitine sensitive and the dominant polymerase of the nucleoplasm probably functions in the synthesis of the many classes of messenger and heterogeneous nuclear RNA1s (111). Polymerase III, a-amanitine resistant and the minor polymerase of the nucleoplasm, i s probably responsible for the synthesis of 4 S RNA and 5 S RNA (112). It has been proposed that through variations in levels and a c t i v i t i e s of the multiple RNA polymerases found in eukaryotes, the synthesis of the major classes of RNA may be regulated. Evidence that changes in the amounts of RNA polymerase may 22. control RNA synthesis comes from a study of changes in the levels and ratios of sea urchin polymerases during early develop-ment (110). The several-fold decline of dRNA synthesis during embryo development was paralleled by a decrease in the amount of chromatographically separated polymerase II. In rat l i v e r , the earliest event in RNA synthesis induced by hydrocortisone i s the stimulation of nucleolar 45 S synthesis (13) . Sajdel and Jacob (114) report that 1 1/2 hours after a single injection of hydrocortisone, a 130-150 percent stimulation in the nucleolar RNA polymerase activity occurs, whereas, the activity of nucleo-plasmic RNA polymerases remain unaffected. Evidence indicates that rRNA accumulation in c e l l s can be controlled at the level of transcription — by the regulation of the synthesis of rRNA precursors. Phytohaemaglutinin-stimulated lymphocytes show an increased rate of synthesis of precursor rRNA when compared to nondividing lymphocytes (115), whereas, the meiotic nucleolus of l i l i e s show a decreased rate of synthesis of rRNA compared to the premeiotic nucleolus (116). Contact inhibited c e l l s exhibit a rate of formation of 18 S and 28 S rRNA some two to four times lower than that shown by exponentially growing c e l l s (117) . The kinetics of synthesis of the precursor to rRNA suggests that the restriction of rRNA formation in contact inhibited c e l l s resides in a reduced rate of transcription of the genes coding ribosomal RNA (117). Transcription of precursor seems to take about 5 min in growing c e l l s but i s extended to some 20 min in contact inhibited c e l l s (118). Just how the rate of transcription i s changed remains a matter for speculation. The actual rate of polymerization of nucleotides by ribosomal RNA polymerase may be controlled, or some step near the termination of transcription may be rate limiting in synthesis. (c) Post-transcriptional Control Mechanisms The amounts of tRNA, rRNA, and mRNA available to the c e l l can be controlled at the transcriptional level. However, superimposed upon this, additional control can be achieved at the post-transcriptional level through regulation of the matura-tion, transport and degradation of these RNA species. The formation of ribosomal RNA i s a model case of a RNA species having a complex multistep maturation process. Our basic understanding of the mammalian process stems largely from the kinetic studies of Perry, Penman, Darnell, and Attardi, together with their respective colleagues (119-122). The primary transcription product arising from nucleolar cistrons i s the 45 S ribosomal precursor RNA. The primary sequence of this 45 S RNA i s altered at the time of transcription by enzyme catalyzed additions of methyl groups to base and sugar moieties. The methylated 4 5 S RNA i s then cleaved to yield smaller com-ponents (i) a 20 S RNA which matures into 18 S RNA; and (ii) a 32 S RNA which matures into 28 S RNA. A small 7 S RNA i s found to be a hydrogen-bonded component of the 28 S RNA and is l i k e l y generated during the f i n a l cleavage of the 32 S precursor molecule (123) . The fingerprint analysis of HeLa c e l l ribosomal RNA and their putative precursors (124) have f u l l y confirmed this proposed maturation pathway for mammalian ribosomal RNA. RNA maturation in the nucleolus occurs concomitantly with ribosome assembly (125,126), 18 S and 28 S RNA emerging from the nucleolus in the form of substantially complete 60 S and 4 0 S ribosomal subunits (127,128) . There i s evidence for the regulation of the fate of rRNA species already transcribed through control of their processing and degradation. For example, hydrocortisone treatment of adrenalectomized rats stimulates 45 S processing (113) , whereas, herpes simplex virus infection retards 45 S processing in human epithelial c e l l s (129) . A reduction in the rate of ribosomal maturation has also been observed in meiotic prophase c e l l s of l i l y microsporocytes (116). Ribosomal RNA accumulation in c e l l s may also be controlled by the turnover of newly synthesized 28 S and 18 S rRNA. For example, Papaconstantinou and Julka (130) found that in mitotically dividing epithelial c e l l s from calf lens tissue, radioactive label could be chased from 45 S through 32 S to 28 S and 18 S rRNA's, whereas , in stationary epithelial c e l l s , no radioactivity appeared in 28 S and 18 S rRNA. Emerson (117) found that in exponentially growing skin fibroblasts the rate of formation of 28 S and 18 S rRNA equaled the rate of net accumulation of rRNA, whereas, in contact inhibited skin fibroblasts the rates of formation of 18 S and 28 S rRNA were about 3 times greater than the rate of net accumulation of rRNA. Emerson concluded that newly synthesized rRNA i s stable in exponentially growing c e l l s , whereas, some fraction of i t i s un-stable and hence degraded in contact inhibited c e l l s . In addition, Cooper (131) has presented evidence to indicate that a fraction of newly synthesized 18 S and 28 S rRNA is degraded in non-growing lymphocytes, this degradation being quickly diminished on addition of phytohaemaglutinin which stimulates lymphocytes to enlarge and divide. Thus, i t seems that cellular regulation mechanisms determine the rate of maturation of the 4 5 S pre-cursors into 18 S and 28 S rRNA and the rate of turnover of these newly synthesized RNA. Transfer RNA i s another RNA species having a complex multi-step maturation process involving both cleavage and base modifica-tion. A precursor species of tRNA appears in the cytoplasm of mammalian c e l l s within a few minutes of i t s transcription in the nucleus (132-134). Gel f i l t r a t i o n studies using Sephadex G-100 (135,136) or electrophoresis on polyacrylamide gels (134) have indicated that this precursor species i s a longer poly-nucleotide than tRNA i t s e l f , by 20-3 0 nucleotides. Altman (137,138) has also isolated tyrosine precursor molecules from E. c o l i infected with J38 0phage carrying a mutant tyrosine tRNA gene and found that they contain 44 extra nucleotides and l i t t l e i f any base modification. Forty-one additional nucleotides were found linked to the 5*-terminus and 3 additional nucleotides were found linked to the common -CCA sequence at the 3'-terminus. The presence of the -CCA sequence at the 3'-end of the precursor molecules suggest that this t r i p l e t i s part of the transcription unit, in contrast to earlier published work (139) . The precursor molecule of wild type tyrosine tRNA appears to be a longer molecule but differs from the mutant precursor only at 3 *-end (138) . The significance of the removal of the terminal sequences of tRNA is not yet clear but i t may serve to control the level of tRNA in the c e l l . Nucleotide modification proved to be unnecessary for in vitro cleavage of the precursors of #80 tyrosine tRNA (138) or of mammalian tRNA (14 0). Because base modification i s probably important in the functioning of the individual tRNA in the translational machinery rather than in the biosynthesis of tRNA i t s e l f , i t w i l l be discussed in relation to translational control. There i s strong evidence to indicate that in eukaryotes, mRNA's also arise as a result of processing a precursor of larger molecular weight. Two recent experiments (141,142) have clearly established that heterogeneous RNA's in the nucleus of eukaryotes, having molecular weights in the range of 2 to 10 million and a base composition similar to that of the specific DNA, are the precursors to cytoplasmic mRNA's. Melli and Pemberton (141) found that approximately 10 % of the heavier fraction of RNA and 20 % of the lighter fraction of RNA isolated from anaemic duck erythrocytes i s complementary to the antimessenger RNA of hemoglobin mRNA (10 S RNA). Stevens and Williamson (142) showed that heterogeneous RNA isolated from the nucleus of myeloma ce l l s i s translated into immunoglobin heavy and light chains when injected into eggs or oocytes of Xenopus laevis. However, the complex set of steps converting these large precursors of mRNA into polysomal forms of mRNA have not as yet been elucidated. Sequences of poly(A), approximately 200 nucleotides long, have been found to be part of the mRNA and heterogeneous nuclear RNA of eukaryotic c e l l s (143-146). The poly(A) located at the 3'-OH end of the RNA molecules (143,147-150) i s added to the Hn RNA post-transcriptionally (151). A l l the mRNA's of mouse L c e l l s , with the exception of those coding for histone, contain poly(A) whereas only one f i f t h of HnRNA of mouse L ce l l s contain poly(A) (152). The low proportion of HnRNA containing poly(A) implies either that polyadenylation i s an event which occurs late in the post-transcriptional processing of HnRNA, or that many HnRNA molecules are not substrates for polyadenylation and therefore cannot be processed into mRNA. When the synthesis of poly(A) i s inhibited in c e l l s infected with adenovirus, addition of poly(A) to adenovirus RNA and transport of mRNA to the cytoplasm are concomitantly reduced (153) . This suggests that poly(A) i s involved in nuclear processing of mRNA sequences and in transport of mRNA from the nucleus to the cytoplasm. However, because mRNA1s coding for histones lack poly(A) (154), poly(A) i s not an absolute requirement for the transport of mRNA from the nucleus to the cytoplasm, nor for engaging into func-tional polyribosomes. On the other hand, because histone mRNA's li k e l y have a metabolic lifetime which i s less than other mRNA's (155), the presence of a poly(A) segment at the 3'-OH end of a mRNA may have a role in determining i t s s t a b i l i t y and therefore, in controlling i t s translation. The observation that in vaccina virus, which replicates in the cytoplasm or in enucleated c e l l s 28 . (156), the RNA, synthesized by RNA polymerase associated with the virion, contains poly(A) at the 3'-terminus (143) suggests poly(A) may play a role in cytoplasmic mRNA function. Similarly, the presence of poly(A) in genomes of single-stranded RNA viruses that serve as mRNA (positive-strand viruses) in infected c e l l s and the absence of such a region from the genome of single-stranded RNA viruses that do not serve as mRNA (negative-stranded viruses) (157) suggest also a role for poly(A) in cytoplasmic mRNA st a b i l i t y and/or translation. However, more data i s needed to define the function of poly(A), i.e., whether i t i s required for the processing to mRNA, for the transport of mRNA from the nucleus, to confer s t a b i l i t y to mRNA or to allow attachment of mRNA to ribosomes. (d) Translational Control Mechanisms Evidence indicates that eukaryotic c e l l s are able to stabilize mRNA's and exert precisely timed selective control over their translation. For example, experiments using actin-omycin D have indicated that in many differentiating systems mRNA is synthesized much earlier than i t s product (a specific protein) i s found in the c e l l . In vivo experiments with Tenebrio  molitor using actinomycin D (158) suggested that in pupa at least part of the mRNA for adult cuticular protein is present on the f i r s t day of pupation and translated five to seven days later. Also experiments with roosters using actinomycin D (159) indicate that estradiol induces the synthesis of mRNA for phosvitin which is not translated u n t i l 20 hours after i t s synthesis has been completed. RNA for several enzymes of slime mold development is stable and made several hours in advance of i t s translation (160). Also the "maternal" or "masked" RNA's synthesized during oogenesis (161), some of which code for microtubular proteins (162), are stored in the egg cytoplasm for several days or weeks not to be translated u n t i l after f e r t i l -ization. The data of Tomkins et a l . (163,164) concerning pat-terns of hormone-dependent induction or repression have led also to a general postulate of modulation of messenger expres-sion after i t s synthesis. The mechanisms by which eukaryotic c e l l s exert selective control over messenger translation have not been elucidated. The s t a b i l i t y or av a i l a b i l i t y of mRNA may be controlled by the binding of certain structural proteins, or specific repres-sors, or by the presence of polynucleotide sequences that are later removed. Correspondingly, some factor could be absent from the c e l l ' s protein synthesizing system and this could limit the c e l l ' s translation of specific mRNA. The adenylation of "maternal" mRNA's may play a role in their activation at f e r t i l i z a t i o n . Slater et a l . (165) hybridized cytoplasmic RNA from unfertilized and four-cell stage sea urchins with [3H]-poly(U) under stringent reaction conditions and demon-strated that the poly(A) t i t r e characteristic of the unfertilized egg increases two-fold between f e r t i l i z a t i o n and the two-cell 30. stage. On the basis of the electrophoretic mobilities of hemo-globin poly(A) sequences and v i r a l RNA's possessing poly(A) tracts of known molecular weights, they estimated the mean composi-tion of unfertilized egg RNA poly(A) sequences to be approxi-mately 100 nucleotides and the mean composition of four c e l l stage RNA poly(A) sequences to be approximately 200 nucleotides. They suggest that f e r t i l i z a t i o n activates pre-existing enzymes for poly(A) synthesis, allowing adenylation of pre-existing mRNA *s to a degree which may be c r i t i c a l for message capacitation. Recent experiments indicate that the secondary structure of mRNA may play a role in the regulation of cistron expression. For example, Fukami et a l . (166) found that R17 bacteriophage RNA preheated in the presence of Mg + + synthesized different amounts of the three R17 proteins in a cell-free system from E. c o l i than R17 RNA preheated in the absence of Mg + +. Because the amounts of the three R17 proteins synthesized could be reversed by appropriate treatment of the RNA, the change in translation frequency of cistrons was not due to degradation of R17 RNA nor to denaturation of a contaminating repressor, but rather to a change in the conformation of R17 RNA. Initiation of synthesis of both the maturation protein and the coat protein of the f2 phage occurs readily in vitro, but i n i t i a t i o n of the RNA poly-merase requires translation of at least part of the coat protein (167) . It seems that the i n i t i a t i o n site of the polymerase cistron becomes unfolded only as the coat cistron i s translated. However, i f f2 RNA is f i r s t treated with formaldehyde, which destroys part of i t s secondary structure and then used to prime synthesis of the three f2 proteins at higher incubation temp-eratures, more polymerase protein i s initiated than usual (167) . As the secondary structure of f2 RNA i s loosened, the i n i t i a t o r sequence of the polymerase cistron is more exposed and allows this cistron to be more easily translated. The nucleotide sequences around the i n i t i a t i o n sites of the three cistrons of phage R17 (168) indicate that no sequence i s common to a l l three of the fragments adjacent to the AUG i n i t -iation codons. Because the three R17 phage proteins are not made in equimolar amounts in infected bacteria or c e l l free systems, the 30 S ribosome subunit of E. c o l i must be able to discrim-inate between the three signals. Differences in the a f f i n i t i e s of i n i t i a t i o n factors for different sequences around i n i t i a t o r codons could provide a mechanism for translational control of the rate of protein synthesis. There i s evidence that in prokaryotes, i n i t i a t i o n factor IF-3, may have a role in determining which mRNA a ribosome can recognize. Two groups of scientists (169-171) have isolated from normal E. c o l i two homogeneous IF-3 factors, one which promotes the recognition of MS2 i n i t i a t i o n sites while the other promotes the recognition of T4 messenger sites. Crude factors prepared from actively growing E. c o l i harvested rapidly in the logarithmic phase usually exhibit good MS2 RNA translation whereas crude factors from stationary phase E. c o l i are much less active for MS2 RNA than for T4 mRNA (172). These variations in uninfected E. c o l i c e l l s are reminiscent of the situation observed after infection by a phage such as T4 where one obtains crude factors which appear devoid of IF-3 activity for MS2 RNA while containg IF-3 activity for T4 mRNA (171). However, when crude factors from both uninfected and T4 infected E. c o l i were fractionated on DEAE-cellulose the IF-3 activity for MS2 RNA could be shown to be present and could be recovered during purification (172). It now appears that an additional protein factor, designated interference factor or factor i , isolated from uninfected E. c o l i i s influencing IF-3 i n i t i a t i o n a c t i v i t y (172,173). When factor i binds to IF-3, i t inhibits in vitro the i n i t i a t i o n and translation of native MS2 RNA while that of T4 mRNA remains active. Also, whereas in vitro the i n i t i a t i o n of translation of MS2 or QB coat protein is inhib-ited by factor i , the synthetase cistron i s more rapidly i n i t -iated in the presence of factor i . Factor i also stimulated in vitro the translation of some phage T4 cistrons while inhib-iti n g the translation of others. Factor i is also one of the three polypeptide chains specified by the host E. c o l i genome, which together with a chain specified by QB RNA synthetase cistron comprise the QB RNA replicase (174). Apparently factor i in vivo f i r s t promotes the translation of the synthetase cistron of an RNA phage and then becomes incorporated in the replicase molecule thereby creating an autoregulatory system. However, a controversy exists as to whether in eukaryotes i n i t i a t i o n factors have a role in determining which mRNA1s a ribosome can recognize. The results of two studies (175,176) strongly suggest that specific i n i t i a t i o n factors may exist in eukaryotes and determine which messengers can be translated by a c e l l . Heywood (175) found that myosin mRNA from chick embryo muscle could function on washed reticulocyte ribosomes, only i f muscle (not reticulocyte) i n i t i a t i o n factors were added. He also found that muscle ribosomes with i n i t i a t i o n factors from reticulocytes could not translate myosin mRNA. At least for myosin mRNA, tissue-specific i n i t i a t i o n factors seem required for i t s translation. Ilan and Ilan (17 6) found that during Tenebrio development, i n i t i a t i o n factors are stage-specific and promote formation of the 8 0 S i n i t i a t i o n complex only with mRNA extracted from the same stage of development. For example, when mRNA was taken from 7-day pupae and i n i t i a t i o n factors from larvae and vice versa, no 80 S i n i t i a t i o n complex was formed. On the other hand, a complete i n i t i a t i o n complex was formed when both mRNA and i n i t i a t i o n factors from the same stage of development were used. Thus, during Tenebrio development, stage-specific i n i t i a t i o n factors may regulate the translation of mRNA. In contrast to the studies above, many recent studies have indicated that neither tissue-specific nor species-specific i n i t i a t i o n factors are obligatory for translation of eukaryotic mRNA's. Lens mRNA's were translated in Krebs II ascites c e l l system with no requirement for additional lens components (177). Similarly, lens (178) and myeloma (179) mRNA's have been trans-lated in a reticulocyte cell-free system, and globin mRNA in Xenopus oocytes (180) and in cell-free extracts from Krebs II ascites c e l l s (181). At least for lens, myeloma and globin mRNA's, neither tissue-specific nor species-specific i n i t i a t i o n factors seems to be required for their translation. Recent experiments indicate that factors inside the human reticulocyte may influence the selective translation of 8 globin mRNA. 0-thalassaemia i s a hereditary anaemia in which the 8-chain of hemoglobin, though structurally normal, i s synthesized abnormally slowly (182-184). The 8-thalassaemia of the Ferrara region of Italy i s distinguished in the homozygote by 8-chain synthesis which i s not merely reduced but completely absent (185,186). In a cell-free system, ribosomes from such subjects do not make 8-chains in the presence of their own supernatant but do when nonthalassaemic supernatant i s substi-tuted (187). Because this effect was due to a factor other than mRNA (187), Ferrara 8-thalassaemia presumably results in a defect in a translation factor. The chain elongation process of protein synthesis can be fac i l i t a t e d in a nonspecific manner by increasing the activ i t y of chain elongation factors. Girgis and Nicholls (188) indicated that the increased rate of protein synthesis observed in livers and kidneys of nephrotic rats may be caused by the increased activity of the chain elongation factor, EF-1, found in these organs. Among the components necessary for code translation, tRNA is the most l i k e l y to be involved in specific translation con-t r o l at the level of chain elongation because of i t s unique feature of being multiple for each amino acid (189-192) and yet ubiquitous for the synthesis of various proteins of the c e l l . The pos s i b i l i t y that the rate of synthesis of certain proteins may be regulated by the concentration of certain species of aminoacyl-tRNA and the relative frequency of the correspond-ing codons in mRNA has been considered on many occasions (188-196). Anderson (197) has demonstrated that the concentration of a tRNA species can regulate the rate of translation of mRNA in vi t r o . The rate of poly (U)-directed [ 1 **C] -phenylalanine incorporation into proteins i s stimulated by increasing concen-trations of tRNA P h e from 1.5 x 10~8M to 3.0 x 10~6M. Similarly, the rate of poly(A,G) directed polypeptide synthesis was directly proportional to the amount of tRNA A r g corresponding to AGA and AGG in the system (197); indicating that E. c o l i tRNA contained only a rate-limiting amount of tRNA A r g specific for AGA and AGG. Anderson and Gilbert (198,199), using a tRNA-dependent rabbit reticulocyte cell-free system, found that when using excess unfractionated tRNA to supplement the system, equal amounts of a and 3 chains of hemoglobin were produced. However, when fractionated tRNA (with a specific portion deleted) was added to the cell-free system, a chain production was reduced to approximately 67 percent the production of 8 chains. Since the rate of translation of a specific natural mRNA can be dif f e r e n t i a l l y slowed in vitro by the simple manipulation of delet-ing a tRNA fraction, then i t i s theoretically possible that a similar mechanism might occur in the c e l l by "turning off" a tRNA genome or by inactivating a previously functioning tRNA. Alternatively, by the activation of a previously synthesized tRNA or by "turning on" a tRNA genome at a specific time, the translation of pre-existing mRNA could be selectively controlled. Indication that such a mechanism does occur during develop-ment, was clearly demonstrated by the recent experiments of Ilan with the mealworm Tenebrio molitor (200-202). As mentioned before, experiments in vivo with Tenebrio pupae using actinomycin D (158),suggest that in pupae, at least part of the messenger for adult cuticular protein i s present on the f i r s t day of pupation and i s translated 5 to 7 days later. Ilan found that both the tRNA fraction and the activating enzyme fraction from 7th day pupae were needed for translation in vitro of mRNA for adult cuticular protein found on 1st or 7th day ribosomes. The tRNA fraction and activating enzyme fraction from 1st day pupae did not function to translate the mRNA for adult cuticular protein on 1st day or 7th day ribosomes. Ilan also found that i f tRNA and enzyme were provided from 7th day pupae which had been treated with dodecylmethyl ether, a juvenile hormone analog which brings about a second pupal molt, pupal cuticle synthesis occurs rather than the typical adult cuticle synthesis. Thus i t seems that 1st day pupae and pupae treated with juvenile hormone lack certain specie(s) of tRNA and aminoacyl-tRNA syn-thetase, this deficiency delaying the translation of adult cuticle mRNA already present in the c e l l . Juvenile hormone seems to act directly or indirectly blocking the synthesis of these "rare aminoacyl-tRNA(s)" needed for adult cuticle mRNA translation. Further experiments by Ilan indicate normal 7th day pupae contain a new leucyl-tRNA synthetase activity and new leucyl-tRNA acceptor activity not found in 1st day or juvenile hormone treated pupae. Although Ilan has found no direct evidence to link the appearance of the new tRNA L e u and i t s synthetase with the switch in protein synthesis, he suggests there i s indirect evidence. The correlation in the timing of the appearance of a new leucine acceptor activity and i t s synthetase, as well as the fact that an enzyme and tRNA are required for the switch in protein synthesis suggests the existence of a link between these two events. Post-transcriptional modification of tRNA may also control the functioning of tRNA at the translational level in the c e l l . Evidence indicates that the degree of modification of a base adjacent to the anticodon strongly influences the function of a tRNA species. The nucleoside, ms 2i 6A, 2-methylthio-6-(A 2-isopentenyl) adenosine, occurs in a position adjacent the 3'-OH end of the anticodon of E. c o l i tRNA T y r (203) . After the infec-tion of E . c o l i with J38 0dsu* i ; [ (a phage carrying the structural gene for suppressor tyrosine tRNA) two new unmodified forms of tRNA T y r are detected (204). Fully modified tRNA T y r, con-taining ms 2i 6A, i s in vitro the most active in protein synthesis and ribosome binding. The tRNA T y r, containing only i 6A, i s one half as active in protein synthesis and ribosome binding activity, while tRNA T y r, containing unmodified A has only 10 percent act i v i t y in protein synthesis and ribosome binding ac t i v i t y . There i s also evidence from another study (205) that the base Y, adjacent the 3'-OH end of the anticodon of Phe tRNA , xs modified during senescence of wheat, disallowing Phe . . . . tRNA participation m protein synthesis. Base methylation seems to be required for certain functions of a tRNA molecule. Aminoacylation of undermethylated E . c o l i Phe tRNA with the appropriate synthetase i s severely restricted. However, aminoacylation can be restored to i t s normal level i f the tRNA in question was remethylated in vitro by specific E . c o l i tRNA methylases (206) . The leucyl-tRNA of E . c o l i , in the absence of methylated bases have codon-recognition activity for only poly(U,C), whereas f u l l y methylated tRNA exhibits code-word response to both poly(U,G) and poly (U,C) (209). It seems the process of tRNA methylation modifies some of the species of leucine tRNA so that they acquire recognition for UG codewords. In another study the coding response of methyl-Phe deficient and normal tRNA were compared. Littauer et a l . (208) f i r s t reported that the methyl-deficient species exhibited ambiguity in codon response but later withdrew this conclusion Phe (209) and reported that methyl-deficient tRNA was bound to Phe the mRNA-ribosome complex less ef f i c i e n t l y than normal tRNA but exhibited the same qualitative response as normal tRNA. Phe This marked reduction in binding of methyl-deficient tRNA to a polynucleotide-ribosome complex may arise from the lack of the 2-methylthio group on the 6-(A 2-isopentenyl) adenosine Phe adjacent to the 3'-OH end of the anticodon of tRNA The cessation of host protein synthesis occurring very early during T-even phage infection seems due in part to the modification of a pre-existing tRNA. It seems T-even phage infection induces a specific ribonuclease that cleaves tRNAL^u at a single phosphodiester bond thereby giving rise to two fragments of approximately equal size (210). This cleavage of tRNA L® u occurs at the site of translation on the hosts polysomes, eliminating about half of the tRNA _. molecules of E. c o l i within 2-3 minutes after infection (211) . Although i t has not been rigorously proven, i t i s l i k e l y that this inac-tivation of tRNA _. i s one of the factors inhibiting host protein synthesis. Since phage mRNA u t i l i z e s l i t t l e , i f any, CUG codons (212) corresponding to codon of tRNAL^u(213-215) i t s translation i s unimpaired. Another type of modification, the removal of the terminal nucleotides ( -pCpCpA) of tRNA1s, is known to affect the functioning of tRNA. If one or more of these terminal nucleo-tides i s removed, the tRNA loses i t s a b i l i t y to accept amino acids (216) and to bind to ribosomes (217) — two processes that are essential for protein synthesis. The major portion of tRNA from non-lactating bovine mammary glands appears to lack one or more of the nucleotides of the terminal -pCpCpA sequence, accounting for the 60 percent decrease in i t s capacity for accepting amino acids compared to lactating bovine mammary gland tRNA, the major portion of which contains the -pCpCpA end (218) . The lack of the -pCpCpA ends seems due to the presence of an active nuclease present in non-lactating mammary glands and not due to the absence of an enzyme that incorpor-ated A and C into the terminal position of tRNA. Thus with the i n i t i a t i o n of lactation, a decrease in nuclease acti v i t y seems to occur, allowing the accumulation of tRNA's with -pCpCpA ends and hence maybe an enhanced rate of protein synthesis. In one case, at least, there i s indication that post-transcriptional modification of tRNA's may control an enzyme's activi t y during development. White et a l . (219) have observed that during the development of Drosophila melanogaster aspartyl-histindinyl-, asparaginyl-, and tyrosyl-tRNA undergo similar post-transcriptional modification. The relative proportion of earlier eluting forms (£ ) and later eluting forms (% ) of tRNA from reverse phase columns for each of these four amino acids alter at specific stages of development in the same way. For example, the ^  forms progressively decrease from egg to late 3rd instar larva and thereafter increase u n t i l 2 weeks after eclosion. The fi forms undergo the reverse relative change. As only one altered nucleotide was detected between the two forms of tRNA A :p, White et a l . (219) bei ieve that modification of a nucleoside analogous to Q, an unidentified nucleoside which i s found only in the f i r s t position of the anticodon of these four tRNA1s in E. c o l i , i s occurring during Drosophila development. Jacobsen and associates (220,221) have shown that tRNA T y r (equivalent to tRNA^ r (219)) inhibits tryptophan pyrrolase activity in the vermilion mutant of Drosophila and that suppression of this inhibition is accom-plished in the mutant su(s) 2 by preventing the occurrence of this form of tyrosine tRNA. The suppressor locus, therefore, l i k e l y specifies the enzyme which somehow modifies the Q base of this tRNA. It has yet to be demonstrated whether tRNA^*" interacts with wild type tryptophan pyrrolase, but i f i t does, as was suggested by Jacobson and Grell (220), i t is l i k e l y that the relative proportion of tRNA^yr and tRNA^1" may control i t s enzymatic activity during the development of Drosophila. 42. III. Transfer RNA Adaptation for Specialized Protein Synthesis During differentiation of an organ for the synthesis of specialized proteins, there can be a disproportionate increase in the amount of tRNA relative to other species of RNA. For example, a five-fold or greater increase in 4 S RNA was found in both nuclear and cytoplasmic RNA preparations of diethyl-stilbesterol stimulated oviducts (222) . Correspondingly, the amount of functional tRNA, measured by total acceptor activity, present in oviduct whole-cell extracts increased after various periods of diethylstilbesterol administration. No qualitative changes in rRNA (3 0 S and 18 S) or other low molecular weight RNA's in these preparations were found. Similarly, c e l l differentiation which occurs in mammary glands during pregnancy i s characterized by a disproportionate increase in tRNA in relation to total RNA (233) . It i s not known at this time whether this disproportionate increase in amount of tRNA i s the result of increased synthesis of tRNA, decreased degradation of tRNA, or a combination of these processes. There i s a report (224) of an apparent hormone induced stabilization of tRNA, causing a reduced rate of degradation in pea stem tissue after indole-3-acetic acid application, and i t i s possible that a similar effect i s being observed in other hormone-induced differentiation. This disproportionate increase in tRNA seems to reflect a preponderant increase in specific tRNA species adapted for the synthesis of the specialized proteins of these differentiated c e l l s . During the lactation period in the mammary gland, pro-tein synthesis consists mainly of two proteins, casein and 8-lactoglobulin. Casein contains a high proportion of glutamic acid (150 residues) and a low proportion of glycine (27 r e s i -dues) compared to other proteins (225) . Correspondingly, a Glu 78 percent increase in tRNA and a 62 percent decrease in tRNA G l y was observed (226) in lactating mammary tRNA prep-arations in comparison with virginal mammary tRNA preparations. There i s also some indication of an increase in glutamyl-tRNA synthetase activity and a decrease in glycyl-tRNA syn-thetase act i v i t y during lactation. In addition, the in vivo Glu charging level of tRNA was markedly increased during Glu lactation when compared with the level of tRNA found during the dry period of mammary glands. Thus at the period of f u l l lac-Glu tation, the correlation between the concentration of tRNA and tRNAG^y and casein composition i s very l i k e l y to ensure the extraordinarily high rate characteristic of protein syn-thesis in mammary gland during lactation. In male and immature female birds, the hepatic synthesis of phosvitin, a protein with an unusually high content of serine residues (over 5 0 percent) i s induced by estrogen admin-istration (227) . Correspondingly, the total serine acceptance of unfractionated hepatic tRNA isolated from roosters during the i n i t i a l period of phosvitin synthesis was more than 25 percent greater than that of hepatic tRNA from control animals. During this rapid phase of phosvitin synthesis, a marked rela-Ser tive increase was observed in one minor (tRNA^^ ) and in one Ser major (tRNA,.^ )^ peak out of the four seryl-tRNA peaks separated Ser by BD-cellulose chromatography. The elevated tRNA peaks corresponded to UCG- and UC(U,C,A)-specific species as deter-mined by binding to E. c o l i ribosomes (228,229). These changes were f u l l y reversible and the seryl-tRNA profile approached the control pattern on termination of phosvitin synthesis (227) . In addition, Maenpa'a has shown in a recent study (230) that the seryl-tRNA profile from rabbit reticulocytes, a c e l l synthesizing mainly hemoglobin was dissimilar to the l i v e r Ser Ser pattern; the UCX-specific species (tRNA^ and tRNA,.^) being Ser decreased compared to AGU (G) -specif i c fraction (tRNA ). The results suggest that the synthesis of specific seryl-tRNA species may be independently regulated and coordinated with the protein structural gene(s) at transcription. Since i t seems reasonable that the c e l l , for the reason of economy of energy and material would develop a regulation system that permits the adaptation of the synthesis of tRNA to the formation of proteins, comparisons of the content of various aminoacyl-tRNA's from various tissues and organs of the same animal have been made (231,232). The relative pro-portions of the total tRNA's for the various amino acids did not vary significantly among brain, l i v e r , kidney and skeletal muscle (231) . In contrast, Ortwerth (232) found that the relative proportions of the total tRNA's for various amino acids did vary significantly between lens and muscle tissue — tissues having different specialized protein synthetic func-tions. This difference in results might be explained by the fact that organs which synthesize a variety of proteins have a similar nonspecialized tRNA content which reflects a com-bination of these many functions. However, aminoacyl-tRNA populations from tissues specialized for the synthesis of specific proteins, such as lens and muscle, may reflect the amino acid composition of these specialized proteins. The idea was further substantiated by studies of the tRNA populations of reticulocytes (233) , plasma c e l l tumours (234), granulation tissue (235,236), and s i l k glands (237,238). When the aminoacyl-tRNA content of rabbit reticulocytes, c e l l s specialized for the production df hemoglobin, was compared with the tRNA content of rabbit l i v e r , a tissue synthesizing many diverse proteins, the histidine tRNA content was found to increase and the isoleucine tRNA content to decrease sig-nificantly in reticulocytes' tRNA preparations (233). This i s consistent with the abundance of histidine and scarcity of isoleucine in hemoglobin. Similarly, Yang (234) found a definite correlation for seven representative amino acids between the specific accepting a c t i v i t i e s of the tRNA from mouse reticulocytes and the M0PC31C plasma c e l l tumour and the amino acid composition of the two major proteins, hemoglobin and IgF myeloma protein, synthesized respectively by these tissues. The relative amount of proline tRNA extracted from rat granulation tissue, a tissue producing large amounts of collagen which i s a protein composed of 33 percent glycine and 22 percent proline plus hydroxyproline (239) was shown in (235) to be significantly greater than tRNA similarly prepared from rat l i v e r . In an additional study (236) the proline and glycine acceptance of tRNA preparations extracted from 15 day granulation tissue was shown to be approximately 3 0 percent greater than that of tRNA preparations extracted from 6 day granulation tissue. Furthermore, cochromatography of 6 and 15 day glycyl-tRNA on BD-cellulose (236) revealed a significant increase in the relative amount of one of the three glycyl-tRNA fractions extracted from the 15 day granulation tissue. Matsuzaki (237,238) compared the aminoacyl-tRNA content of sil k glands, an organ specialized in the synthesis of fibroin which i s a protein mainly composed of the amino acids glycine, alanine, serine, and tyrosine, with the tRNA content of midgut. The glycyl-, alanyl-, and seryl-tRNA content of s i l k glands were increased in amount relative to those in midgut. Thus, the tRNA content of these specialized tissues seems to be adapted for the translation of proteins characteristic of the particular kind of c e l l . Further studies (240-242) have established a direct cor-relation in c e l l s between the pool of aminoacyl-tRNA's and the composition of proteins synthesized. Yamane (240), study-ing four different bacteria, found that the total aminoacyl-ation of five tRNA's correlated well with the composition of these amino acids in the total protein of the corresponding bacteria. Studies on bovine lens tRNA (241) indicate the existence of an adaptative relationship between the proportion of ten aminoacyl-tRNA*s found in tRNA preparations from the internal cortex of the lens and the composition of these amino acids found in the crystallins — the major group of proteins synthesized by this region of the lens. Similarly, in the silk-gland of Bombyx mori, a linear correlation was found to exist between the amino acid distribution in fibroin and the corresponding tRNA's acylated during the secretion phase (242). This regulation process also concerns the intracellular level of aminoacyl-tRNA synthetases in the s i l k gland because their concentrations were found to be almost proportional to the various tRNA's during the secretion period (243). In recent studies (244,245) of the aminoacyl-tRNA content of the posterior part (secreteur) and middle part (reservoir) of the s i l k gland during development., a quantitative adaptation of the tRNA population to the proteins synthesized (fibroin and sericin, respectively) was shown. Transfer RNA correspond-ing to the four major amino acids predominant in fibroin (glycine, alanine, serine, and tyrosine) were found to repre-sent 2/3 of the total supernatant tRNA population of the secre-teur at the 8th day of development. An analogous situation exists in the reservoir, where the tRNA corresponding to the five major amino acids of sericin (serine, glycine, aspartate, glutamate, and alanine) account for 2/3 of the total super-natant tRNA pool at the 7th day. Further, two groups of tRNA could be distinguished; those like the ones above, which have a role in fibroin and sericin production, reaching a maximum concentration in 7-8 days and those primarily involved in the synthesis of other proteins during the growth period, reaching a maximum in 4-5 days. Thus i t seems l i k e l y that there i s an arrest in synthesis after the 4th day of tRNA species not i n -volved in the synthesis of s i l k proteins; whereas tRNA species used in s i l k protein production continue to be actively pro-duced t i l l the 7th or 8th day. Since the h a l f - l i f e df tRNA in an eukaryote i s approximately 8 0 hours (246), by the 7th to 8th day of s i l k gland development, the tRNA population becomes enriched with species necessary for the efficie n t synthesis of s i l k protein by the natural degradation of the other species In this case at least, regulation of tRNA seems to be accomp-lished by "turning off" the synthesis of aminoacyl-tRNA1s no longer needed for the synthesis of protein. There i s also indication of a parallel but not paired increase in a l l isoacceptor tRNA1s specific for the major amino acids of fibroin (24 3). From the growth period (4th day) to the fibroin secretion period (8th day) the tRNAA^a and tRNAG*y species of the posterior part of the si l k gland increase 8 fold, whereas tRNAA*a, tRNA^ y, and tRNA^ y are specifically enriched 16, 35, and 20 fold, respectively. At the 8th day of s i l k gland development, tRNAA^a and tRNAA^a represent approx imately 62 percent and 34 percent of the total alanine acceptor activity of the posterior part of the s i l k gland and tRNAG^y, and tRNAG^y, and tRNAG^y represent 33 percent, 43 percent, and 19 percent, respectively of the total glycine acceptor activity. Since the major codons for these amino acids in fibroin mRNA are known (247) — GGU and GGA for glycine and GGU for alanine — ribosome-binding studies determining the codons used by glycine and alanine isoacceptor fractions of sil k gland tRNA might indicate i f the ratio of specific iso-acceptor-tRNA1s reflect the frequency of synonymous codons in fibroin mRNA. Also, i t remains to be determined whether thi kind of change in isoaccepting tRNA's is merely a response to differing quantities of particular codons in fibroin mRNA or whether i t serves to modulate the translation of fibroin mRNA after i t s synthesis. In this thesis we have undertaken to investigate changes in the tRNA populations during the maturation of salmon testes. We, therefore, f i r s t devised a method of extraction for testis tRNA which gave good yields of active tRNA with l i t t l e DNA contamination. Then, the amounts of tRNA in testes at various stages of development were determined and the changes in amounts of tRNA correlated with the total RNA decrease that occurs during testis development. Studies (226-230, 232-233, 240-245) have suggested that differentiated c e l l s have developed a regulation system that permits the adaptation of the tRNA pool of a c e l l to the amino acid composition of the proteins synthesized by a c e l l . Now, during salmonoid spermatogenesis, a profound alteration in the acid soluble protein composition of chromatin occurs. The typically somatic-type histones, arginine-lysine rich structural proteins of the chromatin, are replaced by protamines, a class of sperm specific proteins, extremely rich in arginine. The amino acid composition of nuclear proteins change markedly with testis maturation; in trout nuclei the arginine content changed from 12.1* percent in September to 32.5 percent in November-December (24). Because of this marked change in amino acid composition of nuclear proteins during salmon testis maturation, salmon testis tRNA preparations were examined for Values are given in percentage of weight of dried isolated nuclei. quantitative changes in the amounts of specific tRNA's. Further, we tried to correlate quantitative changes in the amounts of specific tRNA (tRNA L y s and tRNA A r g) found during testis maturation with changes in the synthesis of the two acid soluble nuclear proteins — histones and protamines. Studies (227-230,236,243) have indicated that a prepon-derant increase in certain isoaccepting tRNA species may occur with specialization of the c e l l for specific protein synthesis. Thus, the arginine tRNA of testes at the various stages of development was examined on BD-cellulose columns and RPC-5 columns in order to detect i f changes in amounts and/or types of arginine isoacceptors occur with specialization of the testis c e l l for protamine synthesis. MATERIALS AND METHODS I. Chemicals and Instruments (a) Chemicals DEAE-cellulose, standard grade from Schleicher and Schuell, Inc., Keene, New Hampshire and CM-cellulose, CM-23, new fibrous, from Whatman were precycled according to procedures in the Whatman Advanced Ion Exchange Cellulose Information Leaflet 1L2 and stored at -20°. Bio-Gel P-10 and Sephadex G-25 were ob-tained from Bio-Rad Laboratories and Pharmacia, respectively. Reversed phase chromatography system*5 employs Plaskon CTFE, 23 00 powder, from A l l i e d Chemical Corp., Morristown, N.J. as the inert support and Adogen 464 from Ashland Chemical Co., Columbus, Ohio as the water immiscible organic extractant. Radioactive amino acids obtained from New England Nuclear were as follows: L-[U- 1 1*C]-lysine, 24 0 mCi/mmole; L-[U- l l fC]-proline, 219 mCi/mmole; [U- 1 1*C]-glycine, 8 0 mCi/mmole; L-[U- 1 **C]-aspartic acid, 162 mCi/mmole; L-[G- 3H]-arginine, 54 2 mCi/mmole. Radioactive amino acids obtained from Schwarz/ Mann were as follows: L-[U- 1"c]-arginine, 150 mCi/mmole and 316 mCi/mmole; L-[U- 1 "*C]-serine, 112 mCi/mmole. Unlabeled arginine, aspartic acid, glycine, lysine, proline, and serine were obtained from Calbiochem. Common chemicals were obtained commercially and were of reagent grade. N,N'-methylenebisacrylamide, N,N,N',N1-tetra-methylethylenediamine, and 6-mercaptoethanol were purchased from Eastman Organic Chemicals. Acrylamide was purchased from Matheson, Coleman and Bell. ATP was purchased from P-L Biochemicals. To minimize bacterial contamination of solutions, a l l solutions after preparation were f i l t e r e d through a Millipore f i l t e r and stored at -20°. To eliminate ribonucleases which stick to glass, a l l glassware was alkali-washed. Dialysis tubing was handled v/ith polyethylene gloves to prevent i t s contamination with skin nucleases (248) . Dialysis tubing was prepared by heating to 8 0° for 30 min in 0.01 M EDTA, pH 7.5 and then by soaking overnight in fresh 0.01 M EDTA, pH 7.5 (249). It was stored in 0.001 M EDTA, pH 7.5. (b) Instruments A TRI-R tissue homogenizer from TRI-R Instruments, Jamaica New York was used with various sizes of teflon pestles and glas homogenizing tubes. A V i r t i s homogenizer from V i r t i s Company, Inc., Gardiner, New York was used with 5 ml to 30 ml diagonal fluted homogenizing flasks. A Waring Blender, Model 7 02CR, from Waring Products Co. had a glass container of working capacity, 500 ml. Flat plate electrophoresis apparatus was purchased from Savant Instrument, Inc., Hicksville, New York. II. Biological Samples (a) Source of Salmon Tissues Testis and liver samples were obtained during the months of April through to September from sexually maturing male Oncorhynchus tschawytscha (chinook salmon) during their mi-gration along the Fraser River in British Columbia. Mature testes and milt of spawning chinook salmon were obtained in October from the Green River Hatchery, State of Washington, USA. The body length and weight and testis weight were recorded for each f i s h . Samples were immediately frozen over dry ice and then transferred to a -8 0° freezer for storage. Other samples needed for incorporation studies were immediately placed on ice and transported to the laboratory within a two to four hour period. Testes removed from chinook salmon caught along the Fraser River betv/een April 1st and approximately May 7th are defined as stage 1 testes. These testes are very immature, represent-ing only 0.2 to 0.5 percent of the body weight of the salmon. Testes removed from chinook salmon caught along the Fraser River in the middle of July are defined as stage 2 testes and rep-resent 1.5 to 3.0 percent of the body weight of the salmon. Testes removed from chinook salmon caught along the Fraser River between the middle and the end of September are defined as stage 3 testes. Stage 3 testes, from f i s h approximately one month from spawning, represent 8.0 to 12.0 percent of the body weight of the salmon. Fully mature testes removed from spawning chinook salmon captured at the Green River Hatchery were defined as stage 4 testes. The stages of salmon testes are detailed more completely in Results and Discussion section. (b) Histology of Salmon Testes Samples of frozen testes were fixed in formalin, embedded, sectioned, and then stained with hematoxylin and eosin. The average diameters of stained testis c e l l s were determined using an eyepiece micrometer. III. Isolation and Characterization of Salmon Testis Protein (a) Arginine Incorporation into Testis Protein Stage 3 and stage 4 testes were homogenized with a TRI-R tissue homogenizer to obtain whole c e l l s for the [ 1^C]-arginine incorporation study. These testes contain small sturdy ce l l s (spermatids and spermatozoa) which remain unbroken with TRI-R tissue homogenization. However, larger c e l l types (sperm- " atogonia and primary spermatocytes), predominate in stage 1 and stage 2 testes, are broken easily by homogenization with the TRI-R tissue homogenizer or the gentler Potter-Elvehjem tissue homogenizer. Thus, rather than a whole c e l l preparation, a fine scissor mince of stage 1 and stage 2 testis tissue was used in the [ 1^C]-arginine incorporation study. A l l operations prior to incubation were performed at 4°. A l l incubations were performed at 20° in a Metabolyte Water Bath Shaker (New Brunswick Scientific Co., Inc., New Brunswick, N.J.) using shaker speed control setting 7.5. Incubations were started by the addition of [1''C]-arginine at a f i n a l concentration of 0.42 yCi/ml of incubation mixture. A l l incubations were terminated by cooling the incubation mixture to 0° . Stage 1 and stage 2 testis tissue (8.5 g of each) in 3 volumes of TMKS was finely minced with scissors. The resulting scissor mince consisted of pieces of testis tissue approximately 0.2 cm3. The stage 1 mince was incubated with [ 1^C]-arginine (316 mCi/mmole) while the stage 2 mince was incubated with [ 1^C]-arginine (150 mCi/mmole). After 2 h incubation, the minced tissue suspensions were homogenized at 4° using 2 or 3 up and down strokes with the TRI-R tissue homogenizer. The resulting nuclei were pelleted by centrifugation (1,000 x g for 10 min) at 4°. After the supernatant was discarded, the pellet was frozen in a dry ice - acetone bath and stored at -20° . Whole ce l l s were prepared from stage 3 and 4 testes by the procedure described by Ling (6). Stage 3 and stage 4 testis tissue (8.5 g of each) in 3 volumes of TMKS buffer was minced with scissors. Then to separate the testis c e l l s , the minced tissue suspensions were homogenized using two or three up and down strokes with TRI-R tissue homogenizer. This homogenate was strained through 4 layers of cheese-cloth to remove con-nective tissue and any large intact tissue. The ce l l s were pelleted by centrifugation (1,000 x g for 10 min) and then resuspended in 2 volumes of TMKS. Stage 3 and stage 4 c e l l s were both incubated with [ 1*C]-arginine (150 mCi/mmole) for 1 h. After the incubation, the c e l l s were pelleted by cen-trifugation (1,000 x g for 10 min) at 4°. After the super-natant was discarded, the pellet was frozen in a dry ice -acetone bath and stored at - 2 0 ° . (b) Fractionation of Testis Protein (i) Acid Extraction of Testis Protein Acid soluble proteins were extracted from these frozen pellets using the procedure of Ling ( 6 ) . The frozen pellets were suspended in five volumes of cold 0 . 2 M H2SOi», b r i e f l y homogenized by the TRI-R homogenizer and allowed to stand at room temperature for 2 0 min. The acid extracts were collected by centrifugation ( 1 , 0 0 0 x g for 1 0 min) and the sediments re-extracted by the same procedure. The sediments (acid insoluble protein pellet) after two acid extractions were drained, weighed and then stored at - 2 0 ° . The two acid extracts from each sample were pooled, the protein precipitated in each by the addition of 3 volumes of 9 5 % ethanol and stored at - 2 0 ° for 3 h. The precipitates were collected by centrifugation ( 1 , 0 0 0 x g for 1 0 min) and were subjected to second precipitations by redissolving in 0 . 2 M H2SO^ and reprecipitating with ethanol at - 2 0 ° . This second precipitation was performed to ensure complete removal of the contaminating free [ 1 >>C] -arginine not incorporated into protein. The precipitates were collected by centrifugation, washed with 9 5 % ethanol, and dissolved in water. The pH's of these protein sulfate solutions were adjusted with NHi^OH to near neutrality as indicated by pH paper. These protein solutions were applied to columns ( 2 . 5 cm x 1 5 cm) of CM-cellulose (H form). Following extensive washing of the columns with water (400 ml), the basic proteins were eluted from the columns with 0.2 M HCl and then lyophilized to dryness. (ii) Bio-Gel P-10 Chromatography The nuclear basic proteins of salmon testes were separated into histones and protamines by Bio-Gel P-10 chromatography. The basic proteins were dissolved in 2 ml of 0.2 M HCl. Approximately 4 0 A 2 3 0 nm units of this protein solution was chromatographed on a Bio-Gel P-10 column (2.5 cm x 25 cm) previously equilibrated with 0.2 M HCl. The column was eluted with 0.2 M HCl at the rate of 1 ml/min and 2 ml fractions collected were monitored at 23 0 nm. This wavelength was used as there are no aromatic chromophores in protamine and hence no absorbance at 28 0 nm. The radioactivity of each fraction of the Bio-Gel P-10 column was determined by counting a 0.1 ml sample from each tube that had been dried on a f i l t e r paper disc (Whatman 3MM, 2.4 cm). In this research, a l l f i l t e r paper discs were placed in standard 20 ml low-potassium glass counting vials containing 5 ml of s c i n t i l l a t i o n mixture (0.4 % w/v 2,5-diphenyloxazole, 0.005 % w/v p-bis [2-(5-phenyloxazolyl)]-benzene in 100 % toluene) and counted in a Nuclear Chicago, Mark I s c i n t i l l a t i o n counter. The carbon-14 counting efficiency on f i l t e r paper discs was 73 percent. 59. (c) Radioactivity of Testis Protein Fractions (i) Total Acid Insoluble Protein A procedure was developed to ensure that the measured radioactivity of the acid insoluble protein was due totally to [1''C]-arginine incorporation into protein and not to radio-activity of acid precipitable [ 1 **C]-arginyl-tRNA presumably also present in the acid insoluble pellet. The usual procedure used to remove nucleic acid present in a protein solution — incubation in 5 % (w/v) TCA at 90° for 15 min (250) — was not used here because the acid insoluble protein remained.as one lump during this heating procedure. This did not allow molecules in the center of the lump to be exposed to the hydrolyzing agent. Rather, the acid insoluble pellet was dissolved in a weak a l k a l i solution (1.8 M Tris HCl pH 8.0) and l e f t at 37° for 2 h. Sarin and Zamecnik (251) found that exposure to 1.8 M Tris HCl pH 3.0 at 37° for 90 min was at least 98 % effective in removing amino acids from tRNA's studied. Weighed samples (approximately 200 mg) of the acid i n -soluble protein pellets were mixed with 3 0 ml of 1.8 M Tris HCl pH 8.0 and then incubated at 37° for 1 h. Lumps of acid insoluble protein remaining in the samples were completely dispersed by homogenization using the TRI-R tissue homogenizer. Then the protein suspension was incubated at 37° for an additional hour. One ml of these solutions were mixed with an equal volume of 20 % (w/v) TCA. The protein precipitates were collected by centrifugation, washed twice with 2 ml of 10 % (w/v) TCA and dissolved in 0.5 ml of 0.1 N NaOH by heating at 100° for 10 min. Duplicate samples (0.1 ml) of these solutions were dried on f i l t e r paper discs and counted. The presence of 0.1 N NaOH in the sample dried on the f i l t e r paper discs did not affect the carbon-14 counting efficiency. (ii) Total Histone or Total Protamine Fractions of the histone peak or the protamine peak from the Bio-Gel P-10 column were pooled. Duplicate samples (0.1 ml) of the resulting solutions were dried on f i l t e r paper discs and counted. Because 0.1 ml had been taken from each fraction of the Bio-Gel P-10 column for determination of radioactivity, values obtained for the pooled solutions were corrected to take into account the aliquots which had been removed. (d) Polyacrylamide Disc Gel Electrophoresis Acid soluble proteins from salmon testes were charac-terized by electrophoresis in 15 % polyacrylamide gels accord-ing to the procedure of Bonner et a l . (252). A 0.01-0.02 ml sample containing approximately 7 yg of protein in 0.2 M HCl containing 20 % (w/v) sucrose was applied to each gel. Electro-phoresis was carried out at 4 mA per tube for 45 min at room temperature. The gel was stained for 6 h with 1 % Amido Black 10 B (British Drug House) in 7 % (v/v) acetic acid containing 40 % (v/v) ethanol and was then destained by exhaustive washing with 7 % (v/v) acetic acid containing 4 0 % (v/v) ethanol. (e) Protein Content of Testis Tissue TCA-tungstate can precipitate polylysine and polyarginine. TCA alone w i l l not precipitate either of these two polypeptides (3). In salmon testes, the arginine and lysine content of basic proteins i s high. Therefore in order to obtain a quantitative yield of a l l proteins from salmon testes, TCA-tungstate was used as the protein precipitating agent. Five to 10 ml of 0.025 M Tris HCl pH 7.5 was added to 1 g of frozen testis tissue. Stage 2, 3, and 4 testis tissue was homogenized at 4° using 10 up and down strokes with a TRI-R tissue homogenizer. Because stage 1 testes contains much more connective tissue than other stages of testes, i t was homogenized for 1 min at 4° using a V i r t i s homogenizer at setting "high" with a Variac reading of 35. Ten ml of solution containing 10 % (w/v) TCA and 0.5 % (w/v) sodium tungstate; f i n a l pH, 2.0, was added to the homogenate. The mixture was allowed to stand for 10 min in an ice bath and was then centrifuged at 4,000 x g for 2 0 min. The resulting pellet was resuspended in a solution containing 5 % (w/v) TCA and 0.25 % (w/v) sodium tungstate; f i n a l pH, 2.0, and homogenized using 2 or 3 up and down strokes with TRI-R tissue homogenizer. This mixture was heated to 90° for 20 min, cooled to 4°, and then centrifuged at 4, 000 x g for 2 0 min. The pellet v/as washed once with a solution containing 5 % TCA and 0.25 % sodium tungstate; f i n a l pH, 2.0. Four ml of 0.1 N NaOII was added to the washed pellet and then to dissolve the protein this mixture was heated at 90° for 10 rain. Some samples required homogenization during this heating procedure to dissolve the proteins completely. The protein concentration of each of these solutions was determined by the biuret method (253) using a standard solution of crystalline bovine serum albumin (Armour Pharmaceutical Co., Chicago, I l l i n o i s ) as standard. In cloudy solutions, diethyl ether v/as used to extract l i p i d s which interfered with the light absorption in the biuret colour reaction. The biuret reaction was used for protein determination because, unlike the Lowry method, i t is not dependent on ' aromatic amino acids (253) and also because histones contain no tryptophan (254,255) and protamines contain neither tryptophan nor tyrosine (35). (f) Arginine Content of Testis Protein Approximately 1 mg of protein in 1 ml of concentrated HCl was hydrolyzed at 110° for 15 h. The hydrolysate was dried in a vacuum desiccator and then dissolved in 10 ml of 10 % potassium hydroxide. The arginine content of the solution was determined using the improved Sakaguchi reaction (256) . This method incorporates acetylation of the sample prior to reaction with 1-naphthol and potassium hypobromite. I l l . Determination of DNA and RNA Content of Whole Tissue One to two grams of frozen testis or l i v e r tissue in 10 volumes of 0.025 M Tris HCl pH 7.5 was homogenized by procedures described for determination of protein content in testis tissue. One ml of this tissue homogenate was then precipitated with 5 ml of 10 % (w/v) TCA (0°, overnight). The precipitate was washed twice with 0.5 M perchloric acid and then once with an ethanol-ether (3:1) mixture. This precipitate was then hydrolyzed in 0.8 ml of 0.3 M KOH (37°, 18 h). After hydrolysis 0.058 ml of 11.6 N perchloric acid was added to the solution to precipitate the DNA. The precipitate containing the DNA was then washed with 0.2 ml of 0.5 M perchloric .acid. The DNA was then hydrolyzed in 1.0 ml of 0.5 M perchloric acid (100°, 10 min) and determined by the diphenylamine method (257). RNA was determined on the combined supernatants (the KOH-hydrolysate and washings of the DNA pellet) by the orcinol reaction (257). IV. Isolation of Salmon Testis tRNA (a) Whole Cell tRNA Testis tissue has a high DNA content when compared to the other tissues. For example, mature testes of Oncorhynchus keta (chum salmon) contain DNA to the extent of about 7.5 percent of their wet weight (258) . This i s about three times the weight percentage of DNA present in calf thymus (259). For this reason, isolation of tRNA with l i t t l e DNA contamination was essential for the study of tRNA in salmon testes. Transfer RNA was f i r s t extracted from salmon l i v e r and testes by Brunngraber's method (260) involving direct homogeniza tion of the tissue with phenol. The procedure was modified by using a homogenizing buffer containing 0.1 M NaCl rather than 1.0 M NaCl. Deoxyribonucleoproteins are least soluble in solutions of 0.1 M to 0.2 M NaCl (261) and should, after homogenization with phenol and centrifugation, be removed as a pellet in the phenol layer. This procedure was further modified by using a homogenizing buffer and phenol both with pH 4.7 rather than pH 7.5. Using phenol pH 4.7 minimizes the amount of DNA entering the aqueous layer. With phenol pH 6.6 to pH 7.5 considerable DNA contam-inates the aqueous layer (2 62) . A l l tRNA fractions studied in this research were prepared using this pH 4.7 extraction method. The exact procedure i s detailed below. The following operations were conducted in a cold room (0°). Frozen tissue was broken into small pieces with a hammer. One hundred and f i f t y ml of solution of 0.1 M sodium acetate pH 4.7, 0.14 M NaCl, 0.005 M EDTA pH 4.7 and 150 ml of phenol saturated with 0.1 M sodium acetate pH 4.7 (3:1 v/v) was added to each 100 g of tissue. This mixture was homogenized in a Waring Blendor at Variac setting 60 for 30 sec. The homogenate was centrifuged in 250 ml polypropylene bottles at 23,000 x g for 20 min. The upper aqueous phase was removed by suction and l e f t on ice. Seventy-five ml of solution of 0.1 M sodium acetate pH 4.7, 0.14 M NaCl, 0.005 M EDTA pH 4.7 was added to the phenol layer of each bottle, mixed by st i r r i n g and recen-trifuged. The aqueous layer was again removed by suction and combined with the other aqueous fraction. The phenol layers were combined and recentrifuged. The small aqueous layer result-ing from their centrifugation was removed by a pipette and combined with the other aqueous fractions. Equal amounts of the combined aqueous fractions were then poured into 250 ml polypropylene centrifuge bottles, to which 75 ml of phenol saturated with 0.1 M sodium acetate pH 4.7 was added. The bottles were then shaken by hand and centrifuged. The aqueous layers were again removed by suction, phenol layers recombined and recentrifuged. To precipitate the RNA, three volumes of 95 percent ethanol were added to the combined aqueous fractions and the solution stored at -20° overnight. It should be noted that in the precipitation of salmon milt tRNA, a mixture of 0.1 volumes of 20 % (w/v) potassium acetate and 0.01 volume of 1 M MgCl2 was added to the ethanol to ensure precipitation. The precipitate was then centrifuged at 450 x g for 20 min and after decanting the supernatant the tubes were inverted to allow a l l the ethanol to drain from the tightly packed precipitate. The RNA pellet was redissolved in d i s t i l l e d water to give a solution of RNA concentration greater than 1.5 mg/ml. An equal volume of 4 M NaCl was added and the RNA solution was stored at 4° for 3 to 8 h. The precipitate containing the high molecular weight RNA (ribosomal RNA and large mRNA) was separated from the supernatant containing low molecular weight RNA, such as tRNA (263), by centrifugation at 4,000 x g for 2 0 min. In order to ensure a better separation of high and low molecular weight material, the RNA precipitate was redis-solved in water and precipitated again with an equal volume of 4 M NaCl. After this RNA solution was stored overnight at 4°, the precipitate containing highly polymerized RNA was centrifuged at 4,000 x g for 20 min. The combined supernatants containing the low molecular weight RNA were dialyzed at 4° for 5 h against 2 changes of 0.01 M Tris HCl pH 8.0 (3,000 ml). The dialysate was then applied to a column (2.5 cm by 25 cm) of DEAE-cellulose (chloride form) which was pre-equilibrated with 0.01 M Tris HCl pH 8.0. A three l i t e r linear gradient running from 0 to 1.5 M NaCl in 0.01 M Tris HCl pH 8.0 buffer eluted 20 ml fractions off the column. The flow rate was 1 to 4 ml/min. The absorbance of each fraction at 260 nm was recorded. Fractions of the tRNA peak were com-bined and the absorbance at 260 nm of this combined fraction measured. The quantity of tRNA extracted can be determined assuming that 1 mg of tRNA per ml in a similar salt solution has an absorbance of 20. Then, this tRNA fraction was flash evaporated at 3 0°. The concentrated solution was then dialyzed at 4° for 6 h against 3 changes of d i s t i l l e d water (3,000 ml). The dialysate was then lyophilized to dryness. To remove amino acids from the terminal adenosine groups of tRNA, the tRNA of a l l tissue except stage 1 testes was dissolved in 3 0 to 4 0 ml of 1.8 M Tris HCl pH 8.0 and l e f t at 37° for 2 h. Sarin and Zamecnik (251) found that exposure to 1.8 M Tris HCl pH 3.0 at 37° for 90 min was at least 98 percent effective in removing amino acids from tRNA's studied. The amino acids were hydrolyzed from stage 1 testis tRNA by incubating the tRNA in 0.5 M Tris HCl pH 8.8 at 37° for 1 h (264). The tRNA was precipitated by the addition of 0.1 volume of 20 % (w/v) potassium acetate and 2 volumes of cold 95 % ethanol. The mixture was l e f t at -2 0° overnight and then the tRNA precipitate centrifuged at 4,000 x g for 20 min. The precipitate was dissolved in a minimal amount of d i s t i l l e d water and stored frozen at -2 0°. (b) Ribosomal tRNA Ribosomal bound tRNA was extracted from 4 00 g of frozen testes from two stage 2 salmon. The tissue was extracted in 50 g lots. F i f t y grams of frozen testis tissue in 100 ml of precooled TMKS buffer was homogenized at 4° in a Waring Blender at Variac setting of 60 for 15 sec. The homogenate was centrifuged at 14,500 x g for 15 min (0°). The decanted supernatant was then centrifuged in a 40 rotor of the Beckman Model L ultracentrifuge at 40,000 rpm for 2 h (4°). After the resulting supernatant was decanted, the pellets were washed two times with cold TMKS buffer. These pellets were resuspended in 0.1 M sodium acetate pH 4.7, 0.14 M NaCl, and 0.005 M EDTA pH 4.7 using 4 or 5 up and down strokes of the TRI-R tissue homogenizer. An equal volume of phenol saturated with 0.1 M sodium acetate pH 4.7 was mixed with the homogenate and the sample was centrifuged at 23,000 x g for 20 min (4°). The aqueous layer resulting from this centrifugation was removed by suction. To this aqueous layer, 0.01 volume of 1 M MgCl2 and 3 volumes of cold 95 % ethanol were added. The RNA was l e f t to precipitate overnight at -20°. From this step the ribosomal bound tRNA was purified by the same procedure used for the purification of bulk tRNA. V. Assay of Amino Acid Acceptor Activity of tRNA Preparations (a) Preparation of Aminoacyl-tRNA Synthetase The 105,000 x g supernatant (S-100) of salmon li v e r or salmon testis extracts was the source of aminoacyl-tRNA synthetases. The S-100 fraction was prepared by a procedure similar to that used by Rosen et a l . (265). A l l preparative procedures were performed at 4°. Sixteen grams of frozen tissue was placed in an ice-cold TMKS solution. When the tissue was par t i a l l y unfrozen, i t was removed and blotted with f i l t e r paper. The tissue was homogenized in 4 g lots with 2.5 volumes of cold TMKS buffer. The tissue was f i r s t cut into small pieces with scissors and then homogenized with a TRI-R homogenizer at moderate speed using about 4 up and down strokes. The mitochondria, nuclei, and c e l l debris were removed by centrifugation at 15,000 x g for 15 min. The yellow fat layer floating on li v e r preparations was removed using a spatula. The supernatant was then removed and cen-trifuged in a 30 rotor of the Beckman Model L ultracentrifuge for 200 min after i t gained a speed of 30,000 rpm. The S-100 fraction was carefully removed without disturbing the cloudy top layer. While st i r r i n g the S-100 fraction enough streptomycin sulfate (Mann Research) was slowly added to S-100 fraction to give a f i n a l concentration of 10 mg/ml. After 10 min, the precipitate was centrifuged at 6,000 x g for 20 min. The supernatant was decanted and i t s pH adjusted to 7.6 with potassium hydroxide. The supernatant was then treated by one of the two following procedures. (a) The supernatant was dialyzed for 4 h against 2 changes of TMKS buffer (1 l i t e r ) . This procedure was used for li v e r aminoacyl-tRNA synthetase preparations used to assay the amino acid accepting acti v i t y of BD-cellulose column fractions. (b) The supernatant was applied to a Sephadex G-25 column (2.5 x 50 cm) pre-equilibrated with TMKS buffer and then eluted with TMKS buffer at a rate of 1 ml/min. The absorbance at 28 0 nm of each 5 ml fraction was recorded and then the fractions of the f i r s t protein peak pooled. This pooled frac-tion was then concentrated by dialysis against polyethylene glycol (Carbowax 6000, Union Carbide), 30 % by weight in TMKS buffer (266) . This procedure was used for a l l testis aminoacyl-tRNA synthetase preparations and for the liv e r aminoacyl-tRNA synthetase preparations used to prepare aminoacyl-tRNA . Pre-cooled glycerol was added to both of these concen-trated enzyme solutions to give a f i n a l content of 4 0 % (v/v) glycerol and then the enzyme preparation was stored at -20°. (b) Purity of Radioactive Amino Acids The purity of a l l radioactive amino acids was checked by spotting 1 yl samples of the undiluted [^C]-amino acids on a paper chromatogram (Whatman # 40, double acid washed) and developing i t in a solvent system of butanol:acetic acid:water (60:15:25 ml) overnight. The chromatogram was then dried and placed over Kodak Blue Brand Medical X-ray film for 24 h. If more than one spot appeared on a radioautogram for an amino acid, spots in the chromatogram were cut out and counted in a s c i n t i l l a t i o n counter. [1I*C] -amino acids having radiochemical impurities greater than 5 % were not used. The Rf of each [1[*C] -amino acid was also compared with Rf of the corresponding 1 2 [ C]-amino acid in the same solvent system. (c) Assay for Amino Acid Acceptor Activity The assay requires the incubation of tRNA with an excess of [^C]-amino acid and aminoacyl-tRNA synthetase in a buffered ++ system containing Mg , ATP, and EDTA. The tRNA is then precipi-tated on paper discs (271) . After the discs are washed thoroughly to remove the excess [^C]-amino acid and other low molecular weight TCA-soluble components of the enzymatic reaction their radioactivity i s determined. The amino acid acceptor capacity of tRNA preparations was measured using a procedure* reported by Hoskinson and Khorana (272) . The composition of the amino acid mix and the washing procedure was modified here. Incubation mixtures (total volume 0.2 ml) contained a tRNA solution (two volumes), a preparation of synthetase (one volume) and a radioactive amino acid mix (one volume). The composition of the amino acid mix used was sodium cacodylate (pH 7.5, 0.4 M), sodium ATP (10 mM), MgCl2 (40 mM) , sodium EDTA (1.6 mM) , and L - [ 1 **C] -amino acid (20 yCi/5 ml The total amino acid concentration of each of the amino acid mixes was as follows: lysine and proline, 20 yM, arginine, serine, and aspartic acid, 40 yM, and glycine, 50 yM. The L-1 1 "C] -serine mix also contained KC1 (4 0 mM) . The range of unfractionated tRNA concentration used in the incubation mixtures was 0.06 to 0.3 0 absorbance unit (26 0 nm) per 0.2 ml. Con-ditions of incubation (time and concentration of enzyme) were adjusted to give maximal labeling and linearity of amino acid acceptance with tRNA concentration. Mixtures were incubated at room temperature (23°) for periods of 2 0 to 60 min. To terminate the reaction, a sample (50 or 100 yl) was withdrawn onto f i l t e r paper discs (Whatman 3MM, 2.4 cm diameter) and plunged into an ice-cold solution of TCA (10 % w/v). The f i l t e r s * Technical Brochure 66 TRI from Schwarz Bioresearch Inc., Orangeburg, N.Y. were then passed through the following series of ice-cold solutions, being allowed to stand at least 15 min in each solution: 10 % w/v TCA, 5% w/v TCA, 95 % ethanol and then ether. Each bath contained 10 ml of solvent per paper disc except the ether bath which contained 3 to 5 ml of solvent per disc. The f i l t e r s were dried under an infra-red light before being placed in vials with 5 ml of toluene s c i n t i l l a t i o n f l u i d (0.4 % w/v 2,5-diphenyloxazole, 0.005 % w.v p-bis [2— (5 — phenyloxazolyl)]-benzene in 100 % toluene). Discs could be removed and vi a l s containing toluene s c i n t i l l a t i o n f l u i d reused several times. Radioactivity was determined in a Nuclear Chicago, Mark I s c i n t i l l a t i o n counter. The counts were corrected for the values obtained for the control to which no RNA was added. The counter was calibrated with samples of radioactive amino acids dried onto paper discs. Column fractions were diluted before assaying i f salt concentrations similar to that of the column fraction were found to inhibit the formation of aminoacyl-tRNA. Since 4.8% (v/v) ethanol was found to inhibit aminoacyl-tRNA formation, ethanol was evaporated at 3 0° from the column fractions using a Rotary Evapo-Mix (Buchler Instruments,Fort Lee, N.J.) before they were assayed. (d) Assay for True Arginine Acceptor Activity Arginyl-tRNA protein transferase i s a soluble enzyme found in the cytoplasmic fraction of mammalian ce l l s v/hich specifically catalyzes the transfer of arginine from arginyl-tRNA into peptide linkage with the amino terminal amino acid of acceptor proteins (273-277) . The arginine transfer reaction i s highly specific with respect to protein acceptors. This spec i f i c i t y i s based on the presence of aspartic acid or glutamic acid at the amino termini (278) . This enzymatic transfer differs from that accompanying the synthesis of proteins de novo in not requiring ribosomes, magnesium ions, or GTP and not being i n -hibited by puromycin. The crude cytoplasmic fraction of salmon li v e r or testis c e l l s used as a source of arginyl-tRNA synthetase enzymes could also contain an arginyl-tRNA protein transferase. True arginyl-tRNA formation should not be measured by cold TCA acid-insoluble incorporation, but rather by the "hot TCA labile fraction", the difference between cold and hot TCA acid-insoluble incorporation. Arginyl-tRNA formation was therefore measured by the same procedure as detailed above. However, duplicate samples were withdrawn onto f i l t e r paper discs and the washing procedure modified so that at the 5 % w/v TCA v/ashing step, one paper remained at 0° for 3 0 min while i t s duplicate remained at 90° for 30 min (279). The arginyl-tRNA formation would equal the difference between cold and hot TCA acid-insoluble incorporation minus the values obtained for the control to which no RNA was added. 74 . VI. Fractionation of Salmon Testis Arginyl-tRNA (a) BD-Cellulose Chromatography DEAE-cellulose (capacity, 1.0 mequiv/g, Whatman DE 22) was benzoylated according to the procedure of Gillam et a l . (267) . BD-cellulose was washed extensively with boiling 2 M NaCl, boiling 2 M NaCl in 25 % ethanol, 2 M NaCl in 50 % ethanol and 2 M NaCl (268) rather than with solutions des-cribed in reference (267) . BD-cellulose, used in a l l columns, was ground to a constant size by passing i t through a No. 50 mesh (0.3 mm opening) and then stored in 2 M NaCl containing 0.02 % sodium azide. Columns were packed, washed, loaded and developed generally as described in (269). Transfer RNA's were eluted from the BD-cellulose columns with linear gradients formed from equal volumes of 0.45 M NaCl and 1.0 M NaCl in 0.01 M MgCl 2. To minimize " t a i l i n g " of tRNA on columns, some columns were eluted with linear salt gradients containing 2 % (v/v) N,N'-dimethyl-formamide (270). (b) Reversed-Phase Chromatography Reversed-phase chromatography (RPC) i s a system in which a water immiscible organic extractant i s present as a film on an inert support and an aqueous solution passed through the column develops the chromatograms. RPC-5 chromatographic system (281) employs Plaskon CTFE powder, a polychlorotrifluoroethylene resin as the inert support and Adogen 4 64, a trialkylmethyl-ammonium chloride, with the predominant chain length of the alkyl groups being Cs - Cio, as the water immiscible organic extractant. RPC-5 chromatographic packing was prepared according to method C (281) . Jacketed glass columns maintained at 37 * 0.1° were packed, equilibrated and developed as described (281). The equilibrating solution used for a l l RPC-5 columns was 0.45 M NaCl, 0.01 M MgCl 2, 0.01 M sodium acetate (pH 4.5) and 0.001 M 3-mercaptoethanol. Two to 6 A 2 6 O units of tRNA were fractionated on (0.9 x 12 cm) columns. These columns were developed with 100 ml gradients of linearly increasing concentrations of NaCl. Forty A 2 6 0 units of tRNA were fraction-ated on a (0.9 x 60 cm) column, using a 800 ml gradient from 0.575 M NaCl to 0.650 M NaCl. Four hundred and f i f t y A2eo units of tRNA were fractionated on a (2 x 50 cm) column using a 2 000 ml gradient from 0.575 M NaCl to 0.64 0 M NaCl. A l l these gradients contained 0.01 M MgCl 2, 0.01 M sodium acetate (pH 4.5) and 0.001 M 3-mercaptoethanol. After gradients were completed, the columns were washed with solutions containing 1.5 M NaCl, 0.01 M MgCl 2, 0.01 M sodium'acetate (pH 4.5) and 0.001 M 3-mercaptoethanol to elute residual tRNA. When deacylated tRNA was fractionated by a RPC-5 column, the column fractions were assayed for arginine acceptor act i v i t y . If [1''C]-arginyl-tRNA was applied to a RPC-5 column, 0.1 to 0.2 ml samples of column fractions were dried on f i l t e r paper discs and counted. If both [ 1 ''C] -arginyl-tRNA and [ 3H] -arginyl-tRNA were applied to a RPC-5 column, 0.2 ml samples of column fractions were pipetted into vials containing 5 ml of Aquasol (New England Nuclear). These vials were shaken vigorously and then counted by liquid s c i n t i l l a t i o n techniques using two channels. Appropriate corrections were made for [^C] radio-activity appearing in the [3H] channel. VI1• Codon Recognition of Salmon Testis Arginyl-tRNAs (a) Preparation of Labeled Arginyl-tRNA The reaction mixture contains per m i l l i l i t e r : 100 ymoles sodium cacodylate (pH 7.5), 2.5 ymoles sodium ATP, 10 ymoles MgCl 2, 0.4 ymoles sodium EDTA, 1.0 A 2 6 0 unit of tRNA, 10 nanomoles of labeled arginine and 0.25 ml of salmon l i v e r aminoacyl-tRNA synthetase preparation. Exact enzyme concen-tration and incubation time for maximal charging was determined beforehand on small samples. After incubation of a reaction mixture at room temperature, the labeled arginyl-tRNA was purified by one of the two following methods. Method 1. After addition of an equal volume of 1 M acetate (pH 4.6) to the reaction mixture, an equal volume of water-saturated phenol (10:75) was added and vigorously stirred (280). The aqueous layer, separated after centrifugation at 12,000 x g for 10 min, was re-extracted a second time with water-saturated phenol. The aqueous phase was applied at 4° to a G-25 Sephadex column (2.5 x 41 cm) pre-equilibrated with 0.5 mM potassium cacodylate pH 5.0 and then eluted with this same buffer at a rate of 1 ml/min. The absorbance at 260 nm of each 5 ml fraction was recorded. Samples (0.05 ml) of each column frac-tion were dried on f i l t e r paper discs and counted. Fractions of the f i r s t A 2 6o absorbing peak, which contained [ l l fC]~ arginyl-tRNA were combined and lyophilized and then stored at -20°. The [ 1 **C]-arginyl-tRNA prepared from unfractionated tRNA of stage 3 testes and from tRNA of BD-cellulose fractions was purified by this method and subsequently used in ribosome binding assays. Because more than 4 0 percent of the [ l k C ] -arginine was found to be hydrolyzed off the tRNA during lyoph-i l i z a t i o n and storage, a better method, method 2, was used for the purification of a l l subsequent labeled arginyl-tRNA preparations. Method 2. After incubation, the reaction mixture was applied directly to a miniature DEAE-cellulose column (10 ml bed volume) equilibrated with 0.25 M NaCl, 10 mM MgCl 2, 10 mM sodium acetate (pH 4.5) and 1 mM 3-mercaptoethanol at 4°. The column was then washed with approximately 4 0 ml of the e q u i l i -brating solution. ATP, enzymes, and free amino acids came out in the washing, while aminoacyl-tRNA's were retained in the column. The labeled aminoacyl-tRNA was then eluted with a solution containing 0.9 M NaCl, 10 mM MgCl 2, 10 mM sodium acetate (pH 4.5) and 1 mM S-mercaptoethanol. Samples (0.01 ml) of each 2 ml column fraction were dried on f i l t e r paper discs and counted. Fractions containing labeled arginyl-tRNA were combined and stored frozen at -20°. (b) Concentration of Arginyl-tRNA Solutions Arginyl-tRNA solutions, with volumes greater than 10 ml, were concentrated f i r s t by applying them to miniature DEAE-cellulose columns. These miniature columns were constructed using 2.5 cc disposable plastic syringes, the bottom of which were f i t t e d with small glass wool plugs. DEAE-cellulose was packed to the 1.5 cc mark. These columns were equilibrated with 0.25 M NaCl, 10 mM MgCl2 10 mM sodium acetate (pH 4.5) and 1 mM 3-mercaptoethanol. The tRNA solutions were diluted to NaCl concentrations less than 0.25 M. These diluted tRNA solutions were then applied to the columns and allowed to percolate through unimpeded u n t i l the last drop of solution reached the top of the matrixes. Then the columns were eluted with 0.9 M NaCl, 10 mM MgCl 2, 10 mM sodium acetate (pH 4.5) and 1 mM 8-mercaptoethanol. Four to 6 ml were collected after the f i r s t m i l l i l i t e r . These combined fractions were mixed with 2.5 volumes of 95 % ethanol and stored at -20° overnight. The ethanol solutions were then f i l t e r e d through millipore f i l t e r s (HA Millipore f i l t e r , 13 mm in diameter, 0.45 y pore size). The tRNA precipitate on the f i l t e r s were eluted by putting the f i l t e r into 1 ml disposable conical microcentrifuge tubes with 0.5 ml of 0.01 M sodium acetate (pH 4.5). This was l e f t on ice for one hour and mixed on a vortex occasionally. Then these f i l t e r s were removed and the arginyl-tRNA solutions stored at -20°. By this procedure, 90 percent of the arginyl-tRNA was recovered from solutions with tRNA concentrations not less than 0.1 A 26o/ml. '(c) Preparation and Characterization of Arginine tRNA Codons (i) Synthesis of Trinucleotides Trinucleotides were synthesized from the appropriate dinucleoside monophosphates using primer-requiring polynucleo-tide phosphorylase from Micrococcus lysodeitkticus and nucleo-side 5'-diphosphates. Nucleoside 51-diphosphates (ADP, CDP, GDP, UDP) and dinucleoside monophosphates (ApG and CpG) purchased from P-L Biochemicals, Inc., Millwakee, Wisconsin, and Raylo Chemicals, Ltd., Edmonton, Alberta, were used without further purification. Reaction mixtures for the synthesis of a trimer XpYpN contained 10 mM MgCl 2, 0.4 M NaCl, 0.2 M glycine i buffer (pH 9.3), 1.0 mM NDP, 6 mM XpY, and 0.3 mg/ml poly-nucleotide phosphorylase (P-L Biochemicals, Inc.) in 2 ml to 4 ml total volume (282) . Prior to addition of the enzyme, reaction mixtures were heated to 70° for 5 min and then cooled to 37° (283). After incubation with enzyme at 34° for 4 h, the reaction was terminated by placing the tube in a boiling water bath for 4 min. The reaction mixtures were diluted with 19 volumes of water and then applied to columns (1.2 x 50 cm) of DEAE-cellulose (bicarbonate form). Nucleotides were eluted from the columns at rate of approximately 1.5 ml/min using linearly increasing concentrations of NH^HC03 (pH 8.0) (284). CpGpA, CpGpC, CpGpU were eluted with linear gradients formed from equal volumes (1 1 to 1.8 1) of d i s t i l l e d water and 0.15 M NH!,HC03. ApGpA and CpGpG were eluted with linear gradients formed from 1.4 1 each of d i s t i l l e d water and 0.175 M N I U H C O 3 pH 8.0. ApGpG was eluted with a linear gradient formed from 1.4 1 each of d i s t i l l e d water and 0.20 M NHi»IICO3 pH 8.0. Residual nucleotides remaining on the column were eluted with a solution of 1.0 M NH\HC0 3 pH 8.0. The 10 ml fractions were monitored at 26 0 nm. Fractions containing the trinucleotides were combined and the trinucleotide concentration estimated by measuring their absorbance at 260 nm at pH 7.0. The approp-riate extinction coefficient was calculated by averaging the £ 2 6 0 of the constituent nucleotides and adjusting for hypochromic shift due to base stacking of trinucleotides. The yield, therefore, was calculated as the percentage of nucleoside 5'-diphosphate recovered as trinucleotide diphosphate. The com-bined fractions were then concentrated to dryness in a rotatory evaporator (bath temperature 30°). Residual ammonium carbonate was removed by addition of aqueous ethanol and repeated evap-oration. The residual ammonium salt of the nucleotide was dissolved in a small amount of water to give a solution of approximately 30 A 2 6o units/ml and then stored frozen at -20°. Fractions from the DEAE-cellulose columns containing the d i -nucleoside monophosphate were also combined and concentrated. The purity of these re-isolated dinucleoside monophosphates was tested in two paper chromatography systems (solvents A and B) and used again in subsequent reactions with other nucleoside 5'-diphosphates. (ii) Paper Chromatography and Electrophoresis Chromatography on Whatman #4 0 (double acid washed) paper was carried out by the descending technique at 23°. Solvent systems used were: A n-propanol: concentrated ammonia: water (55:10:35ml)(285) B ammonium sulphate: 0.1 M sodium phosphate pH 7.0 (40g:100mlj (286) C 95 % ethanol: M ammonium acetate pH 7.5 (7:3ml) (287) D isobutyric acid: concentrated ammonia: water pH 3.8 (66:1:33ml) (288) E isobutyric acid: M ammonium hydroxide (5:3ml) (289) F isopropanol: concentrated ammonia: water (7:1:2ml) (290) G n-propanol: concentrated ammonia: water (66:7.5:32.5 ml) In an additional chromatography system, H, the paper (Whatman 3MM) was pre-soaked in 0.2 M sodium phosphate pH 7.4, dried, spotted and developed in solvent F (291) . Analytical electrophoresis, I, for base ratio determinations was performed on a Savant f l a t plate apparatus on Whatman #4 0 paper at pH 3.5 in 0.05 M sodium formate at 60 volts per cm for 1 hour (2 92). Prior to chromatography, a l l samples were heated to 70° for 5 minutes to reduce G-G interactions (293). Nucleotides were detected under short-wavelength ultraviolet using a Chromatovue from Ultraviolet Products Incorporated. ( i i i ) Characterization of Di- and Trinucleotides The purity of each oligonucleotide was estimated by sub-jecting at least 2.0 A 2 6 o units of each to two paper chromato-graphy systems, either to systems A and B or to systems C and D. Chromatographic mobilities in solvents A and B were c a l -culated relative to that of adenosine 5'-phosphate and in sol-vents C and D were calculated relative to that of uridine 3'-phosphate. The limit of visual detection of an ultraviolet absorbing compound after chromatography in this manner i s less than 0.05 A 2 6 o unit; consequently, compounds which migrated as a single component in both chromatography systems were considered at least 97 percent pure. Each compound was also degraded with T 2-ribonuclease and with snake venom phosphodiesterase as previously described (283) in order to confirm i t s identity as the expected product It should be noted that 3.2 yg of T 2-ribonuclease (Calbiochem) and 5 yg of snake venom phosphodiesterase (Worthington) were used in the digestion mixtures. Digestion products were sep-arated by paper chromatography in solvent systems described previously or by electrophoresis at pH 3.5. Each ultraviolet light-absorbing spot was eluted (294) with 0.1 % NR\OH and i t s spectrum was read against appropriately eluted paper blanks. As confirmation of structure, molar base ratios of digestion products were calculated on the basis of the spectro-photometrically determined concentration of the eluate of each spot. (d) Preparation of E. c o l i Ribosomes (i) Growth of E. c o l i  Escherichia c o l i MRE-600 was grown in a medium (pH 6.8) containing 10 g of yeast extract (Difco Laboratories, Detroit, Michigan), 34 g of KH2PCU, 9 g of KOH and 10 g of glucose per l i t e r (294) . A 10 % (w/v) glucose solution was ste r i l i z e d and added to the ste r i l i z e d yeast extract solution after both were cooled. Four flasks, each containing 500 ml of ste r i l i z e d medium, were individually inoculated with 10 ml of E. c o l i MRE-600 grown in broth cultures and l e f t shaking for aeration at 37° for 12 h. These 2 l i t e r s of E. c o l i were used as innoculum for 20 l i t e r s of unsterilized medium in the Biogen (American S t e r i l i z e r Co., Erie, Pennsylvania). Antifoam A (Dow-Corning Corp.) was sprayed on the surface of the medium to prevent foaming during growth of the bacteria. The E. c o l i were grown in the Biogen at 3 6°, with an air pressure of 15 p. s . i . and an agitation setting of 142. After 90 min, the temperature setting was decreased to 10° and ce l l s were har-vested immediately by means of a Sharpless centrifuge. The ce l l s were washed by rapid suspension in 250 ml of 10 mM Tris HCl pH 7.8, 14 mM magnesium acetate, and 60 mM KCl at 4° and were centrifuged in pre-weighed tubes at 15,000 x g for 15 min. The supernatant s o l u t i o n s v;ere decanted and the p e l l e t s were d r a i n e d . C e n t r i f u g e tubes were a g a i n weighed and a y i e l d o f 7.5 g of packed c e l l s , wet weight, per l i t e r o f medium was o b t a i n e d . These c e l l s were f r o z e n i n a dry i c e - a c e t o n e bath and s t o r e d a t -3 0°. ( i i ) I s o l a t i o n o f Ribosomes F o r t y - e i g h t grams of E. c o l i MRE-600 c e l l s were thawed a t 4° and then e v e n l y suspended i n 2 volumes o f f r e s h l y prepared b u f f e r (w/v) c o n t a i n i n g 10 mM T r i s HCl pH 7.8, 14 mM magnesium a c e t a t e , 60 mM KC1 and 6 mM 3-mercaptoethanol. The c e l l s were broken i n a French p r e s s (American Instrument Co., Inc., S i l v e r S p r i n g , Maryland, USA) w i t h 18,000 p . s . i . i n a p r e c h i l l e d c y l i n d e r (296). The e x t r a c t was c e n t r i f u g e d a t 20,000 x g f o r 20 min. The supernatant was removed to w i t h i n 1 cm of the p e l l e t and was c e n t r i f u g e d a t 3 0,000 x g f o r 3 0 min. The supernatant was removed to w i t h i n 1 cm o f the p e l l e t and c e n t r i f u g e d i n a 40 r o t o r o f the Beckman Model L u l t r a c e n t r i f u g e a t 105,000 x g f o r 2 h. The supernatant was decanted and d i s -c a r d e d . The ri b o s o m a l p e l l e t was suspended i n 96 ml of a s o l u t i o n c o n t a i n i n g 2 M K C l , 3 0 mM magnesium a c e t a t e , 6 mM 3-mercaptoethanol and 10 mM T r i s HCl pH 7.8 by g e n t l y homogen-i z a t i o n (four o r f i v e passes) i n a P o t t e r - E l v e h j e m homogenizer and then l e f t on i c e o v e r n i g h t . The r i b o s o m a l suspension was c e n t r i f u g e d a t 105,000 x g f o r 2 h, and then the supernatant s o l u t i o n decanted and d i s c a r d e d . The ribosomes were washed 3 times with the 2 M KCl buffer by gentle suspension and sedi-mentation by centrifugation (297). The washed pellets were suspended in 24 ml of buffer containing 10 mM Tris HCl pH 7.8, 14 mM magnesium acetate, 60 mM KCl, and 6 mM 8-mercaptoethanol and the suspension centrifuged at 10,000 x g for 5 min to remove aggregates (296) . The washed ribosomal fraction was divided into small aliquots, frozen in a dry ice-acetone bath and stored at -80°. (e) Assay of Arginyl-tRNA Binding to Ribosomes The binding of [ 1 **C]-arginyl-tRNA to ribosomes was carried out according to the procedure described by Nirenberg and Leder (2 98). Reaction mixtures contained the following com-ponents in a f i n a l volume of 0.05 ml: standard buffer (0.1 M Tris HCl, pH 7.5, 0.05 M potassium chloride, 0.02 M magnesium acetate), E. c o l i MRE-600 ribosomes (2.4 A 2 6 o units), a t r i -nucleotide (approximately 6.0 nmoles) and [ 1^C]-arginyl-tRNA as specified on each table. The exact amount of a trinucleotide added to a reaction mixture was as follows: ApGpA, 5.87 nmoles, ApGpG, 5.85 nmoles, CpGpA, 5.92 nmoles, CpGpC, 6.7 6 nmoles, CpGpG, 6.27 nmoles, and CpGpU, 5.4 nmoles. The control con-tained a l l components except a trinucleotide. The standard buffer and the trinucleotide were added f i r s t , heated to 70° for 5 min and then the incubation mixture cooled for 5 min at 25°. Ribosomes were added and then the reaction initiated by the addition of [1''C]-arginyl-tRNA. Each reaction mixture was incubated for 20 min at 25°, then diluted with 3 mis of stan-dard buffer (4°) and washed on Millipore f i l t e r s as described (298). F i l t e r s were dried under the infra-red light and counted. A l l binding assays were performed in duplicate. RESULTS AND DISCUSSION I. Study of Spermatogenesis in 0. tschawytscha The study of spermatogenesis in Oncorhynchus tschawytscha was undertaken with the primary objective of defining a system where tissue would be readily available in sufficient quan-t i t i e s to allow the study of tRNA at a l l stages of testis development. 0. tschawytscha was chosen for investigation because individuals are the largest of the species of Pacific salmon and because i t migrates into fresh water, i.e., i s . readily accessible for collection of tissue samples, at a l l stages of sexual maturity. (a) Increase in Testis Size During Spermatogenesis It i s necessary to express the size of the testes in a standard way because of the v a r i a b i l i t y in the size of individual f i s h . The commonly used gonosomatic index which relates gonad weight to total weight i s not appropriate to sexually maturing salmonids which undergo starvation during sexual maturation. Instead, the development of 0. tschawytscha i s expressed in terms of gonad weight per unit length (11,17,299). Changes in the testis weight of 0. tschawytscha during sexual maturation are shown for a fi s h of standard length in figure 1. The dramatic increase in testis size which i s char-acteristic of salmonids undergoing sexual maturation (22,299) also was found with 0. tschawytscha in the present study. Figure 1. Change in the testis weight of sexually maturing 0 . tschawytscha. The data were calculated for a standard f i s h of length 75 cm (the small change in the overall length of the f i s h result ing from changes in head length during maturation was neglected) At each sampling time the relationship between the length of calculated for each individual. The mean value of n for the group was determined and used to calculate the testis weight for a f i s h 75 cm long. The vertical bars on the graph define the standard deviation for each value. Each sample consisted of at least six f i s h . The time scale starts on April 1st. The sample at stage 4 (Green River fish) was in fact collected at approximately the same time as the stage 3 (Fraser River f i s h ) . However, the Fraser River fis h , which were used for a l l samples other than stage 4, mature about 1 month after stage 3. fi s h and testis weight TIME ( W K S ) Testis development takes place over a six month period, i n -volving an increase in testis weight from approximately 20 g to several hundred grams in a 10 kg f i s h . The immature testes (stage 1) represents 0.2 to 0.5 percent of the body weight of the salmon. Stage 2 testes, approximately 1.5 to 3.0 per-cent of the body weight, represent testes in the middle of the four month logarithmic growth phase. Stage 3 testes, from f i s h approximately one month from spawning, are at their maximum mass, a weight representing 8 to 12 percent of the body weight of the salmon. The loss in testis weight during the last stage of maturation could be due to the loss of sperm or because the f i s h sampled at stage 3 and stage 4 were from different stocks. However, care was taken that f i s h sampled at stage 4, did not show signs of free running sperm, i.e., they were probably a few days away from f u l l maturity. Also, because DNA analysis (Table 5) suggest that there was no loss of c e l l s at stage 4, the weight decrease i s more l i k e l y due to a loss of cytoplasm from testes as the sperm undergo spermio-genesis. Loss of testis weight during the f i n a l stage of maturation was also observed in studies on 0. nerka (300,301) and on Salmo gairdnerii (26). k^) Histology of Spermatogenesis The histology of the four stages of 0. tscb.-., ytscha testes is seen in figure 2. Stage 1 testes, which are completely immature testes, consist of closely packed cysts of primitive Figure 2. Histology of developing 0. tschawytscha testes. The four stages of development are defined in figure 1. The approximate magnification of these testis slices i s as follows: 1, x 370; 2, x 330; 3, x 300; and 4, x 190. The predominant c e l l s of stage 1 testes are spermatogonia, with diameter measuring 4 to 6 y. The light staining c e l l s of stage 2 testes are spermatocytes, with diameters measuring 3 to 4 y. The dark staining c e l l s of stage 3 testes are sperm-atids having an average diameter of 2.5 y. The spermatozoa o stage 4 testes have diameters measuring 1.3 to 2.0 y. 9 0 a germ c e l l s (spermatogonia). These cysts are separated by a large amount of connective tissue, making homogenization of this stage of testes very d i f f i c u l t . In stage 2 testes the ce l l s have increased in number by mitosis resulting in larger cysts and smaller germ c e l l s . There i s also a reduction in the relative amount of interlobular connective tissue; the walls of the primitive cysts having largely disappeared. Spermatogonia are no longer seen, having now developed into primary and secondary spermatocytes. Occasional groups of spermatids are also observed in stage 2 testes. Greater than one half of the c e l l s of stage 3 testes consist of spermatids and spermatozoa, the remainder are spermatocytes. Note also the obvious condensation of the spermatid nuclear material into homogeneous dark staining spheres. The contents of the cysts of stage 4 testes consist entirely of spermatozoa. (c) Changes in Testis Proteins During Spermatogenesis To characterize the basic proteins present in 0. tschawytscha testes at the various stages of maturation, acid extractable proteins were examined by polyacrylamide gel electrophoresis (figure 3). The protein band pattern of these gels indicate that histones are present in nuclei of stage 1, 2,and 3 testes, while protamines are present in the nuclei of stage 2, 3, and 4 testes. The band patterns indicate that protamines are absent from stage 1 testes and histones are absent from stage 4 testes. F i g u r e 3. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of the t o t a l a c i d s o l u b l e p r o t e i n s of O . tschawytscha t e s t e s a t v a r i o u s stages of spermatogenesis. +ve -ve H i s t o n e s Protamines A c i d s o l u b l e p r o t e i n s from v a r i o u s stages of salmon t e s t e s were e x t r a c t e d w i t h 0.2 M H 2 S O i , as d e s c r i b e d i n M a t e r i a l s and Methods. Approximately 7 ug of these a c i d s o l u b l e p r o t e i n s i n 0.2 N HCl c o n t a i n i n g 20 % (w/v) sucrose was a p p l i e d to each 15 % g e l and e l e c t r o p h o r e s e d f o r 45 min a t 4 mA per tube. 1, stage 1; 2, stage 2; 3, stage 3; 4, stage 4. Figure 4 shows the Bio-Gel P-10 chromatography of acid extractable proteins from 0. tschawytscha testes at the various stages of maturation. The proteins are eluted from the column as two major peaks, Pj and Pjj- These peaks have previously been characterized by Ingles (19) who has shown by polyacryla-mide gel electrophoresis that P^ . contains mainly histones and other large molecular weight (greater than 10,000) basic pro-teins while Pjj contains only protamine. The Bio-Gel P-10 absorbance profile of stage 1 acid soluble proteins again indicates the absence of protamine in nuclei of stage 1 testes. The profiles of stage 2 and 3 acid soluble proteins indicate the presence of both histones and protamines in the nuclei of these testes. The presence of only one peak ( P J J ) in the acid soluble protein profile of stage 4 testes indicates the presence of only protamine in these nuclei. Thus, both sets of data indicate that during the early stage of spermatogenesis (stage 1) the testis nuclei contain mainly histone and by the end of spermatogenesis (stage 4) the testis nuclei contain only protamine. These results suggest that during spermatogensis of 0. tschawytscha, a replacement of histones by protamines has occurred in the testis c e l l nuclei and support the observation of various investigators who have examined this transformation in developing testes of salmonoid f i s h (8,24,25,30,35). However, there seems to be an earlier appearance of protamine in the testes of naturally maturing chinook salmon than following hormonally induced Figure 4. Transformation from histone synthesis to protamine synthesis in 0. tschawytscha testes. Testis minces or testis c e l l suspensions were incubated with [ 1 **C]-arginine (0.42 yCi/ml incubation mixture) at 20° for 2 h (stage 1 and 2) and 1 h (stage 3 and 4). Acid soluble proteins were extracted with 0*2 M H2S0!, and converted to chloride form by passage through CM-cellulose columns. Acid soluble proteins (41 A 2 3 0 units - stage 1 and 2; 39 A 2 3 0 units - stage 3 and 4) were chromatographed on Bio-Gel P-10 columns (2.5 cm x 25 cm). The column was eluted with 0.2 M HCl at rate of 1 ml/min and 2 ml fractions were collected. 1, stage 1, 22.8 g testes; 2, stage 2, 101 g testes; 3, stage 3, 669 g testes; 4, stage 4, 545 g testes ( a l l weights stan-dardized to 75 cm f i s h ) . Acid soluble proteins are eluted from Bio-Gel P-10 columns as two major peaks, Pj and P J J . The f i r s t peak (P_) eluted i s composed mainly of histones while the second peak iPjj) contains only protamine (6,19). It should be noted, especially in Bio-Gel P-10 column of stage 2 testes, that the radioactive profile of [ 1 **C]-arginine incorporation in protamine i s not coincident with the absorbance profile of protamine because newly synthesized protamine i s more highly phosphorylated and does not elute in the same position as protamine synthesized earlier and already dephosphorylated (6,302). 94a spermatogenesis in rainbow trout (25,30). Acid soluble pro-teins from hormonally induced trout testes in the middle of their logarithmic growth phase (equivalent to stage 2 testes) show no protamine band when electrophoresed on polyacrylamide gels (30) . In contrast, the acid soluble proteins of naturally maturing salmon testes at stage 2 show a detectable protamine band on polyacrylamide gel electrophoresis (figure 3) and a small protamine peak ( P J J ) on Bio-Gel P-10 chromatography (figure 4). Since naturally maturing salmonids are less synchronous in testis development than hormonally injected salmonids (3 03), protamine synthesis begins in less mature testes of naturally maturing f i s h . The relative incorporation of [ 1^C]-arginine into histones and protamines at different stages of testis development i s shown in figure 4 and table 1 & 2. In stage 1 testes, incor-poration of labeled arginine into acid extractable proteins was found mainly in peak Pj, indicating active synthesis of histones. While 39.0% of the arginine incorporated was used to synthesize histones, only 6.8 % was used to synthesize protamine. From table 2 i t can be seen that approximately 28.3 % of the total protein synthesized by stage 1 testes i s histone. In stage 2 testes, incorporation of labeled arginine into histones (Pj) had decreased to 8.9 % and most of the incorporated [11*C] -arginine (73.3 %) appears in protamine (P I ; [) . From values in table 2 i t can be seen that approximately equal amounts of histones and protamines are synthesized by Table 1. In vitro incorporation of [ 1 "*C]-arginine into pro-teins of 0. tschawytscha testes. Stage Acid insoluble Histone Protamine Protein CPM/ga CPM/g % CPM/g % 27,720 54 .2 19,964 39. 0 3,470 6 .8 27,000 17.8 13,467 8. 9 110,962 73 .3 17,750 23.5 2,182 2. 9 55,700 73 .6 4,738 66.3 179 2. 5 2,222 31 .2 Radioactivity incorporated expressed as CPM/g testes (wet weight). Percentage of total arginine incorporation in each protein fraction. Testis minces or testis c e l l suspensions were incubated with [ 1 ''C] -arginine (0.42 yCi/ml incubation mixture) at 20° for 2 h (1 & 2) and 1 h (3 & 4). Acid soluble proteins were extracted with 0.2 M H 2 S O i , j leaving an acid insoluble protein pellet (3 04). The histone fraction was separated from the protamine fraction by chromatography on Bio-Gel P-10. The amount of radioactivity incorporated in total acid insoluble proteins, histones, and protamines was determined as described in Materials and Methods. 1,, stage 1, 22.8 g testes; 2, stage 2, 101 g testes; 3, stage 3, 669 g testes; 4, stage 4, 545 g testes (all values standardized to 75 cm f i s h ) . Table 2. Calculations of percentage of each type of protein synthesized in the four stages of salmon testes. Stage Acid Insoluble Histone Protamine Protamine/Histone Protein % % % 1 71.1 28.3 0.6 0.02 2 64.1 18.1 17.8 0.98 3 78.2 5.4 16.4 3.04 4 95.0 2.0 3.0 1.50 [ 1^C]-arginine incorporation values [CPM/g testes (wet weight)] from Table 1 were normalized to an arginine content of 5 % assuming acid insoluble proteins, histones and protamines have average arginine contents of 4.5 %, 8 %, and 67 %, respec-tively. The normalized values of each protein fraction were then expressed as a percentage of the total protein synthesized. The average arginine content of acid insoluble proteins was estimated from the mean value of arginine in unrelated proteins (305) and mammalian (non-histone) proteins (306). Because the histone profile of a salmonoid testes at early stages of spermatogenesis i s similar to the histone profile of a salmonoid l i v e r (3 0 ), the arginine content ot testis histones was assumed to be 8 %, the value stated by Palau and Butler (3 07) for trout l i v e r histones. Protamines of 0. tschawytscha testes have an arginine content of 67 % (19). stage 2 testes. Incorporation of labeled arginine into histones has v i r t u a l l y ceased (2.9 %) in stage 3 testes while 73.6 % of the labeled arginine i s incorporated into protamine. Approx-imately 16.4 % of the total protein synthesized by stage 3 testes i s protamine while only approximately 5.4 % i s histone. In stage 4 testes, the amount of labeled arginine incorporated into protamine decreased to 31.2 % and accounts for only approximately 3.0 % of the total protein synthesized. In addition, Ling et a l . (3 04) found that mature sperm of rainbow trout (Salmo gairdnerii) did not incorporate [1''C]-arginine into protamine. Recent experiments by Louie and Dixon (26) have elearly shown that histone and DNA synthesis in rainbow trout testes occur in the spermatogonia and in the primary spermatocytes. Since stage 1 salmon testes consist mainly of spermatogonia, their active synthesis of histones i s expected. Louie and Dixon (26) in accord with histochemical observations of Alfert (8) have shown that protamine synthesis occurs during the maturation of spermatids. Since stage 2 salmon testes consists of both spermatocytes and spermatids, i t s synthesis of approx-imately equal amounts of protamine and histone i s also expected. It has been observed in several systems that DNA and histone synthesis are closely coupled (308 - 310). Since by stage 3 most testis c e l l s have ceased dividing and therefore also DNA syn-thesis, histone synthesis has v i r t u a l l y ceased in this stage testes. Then, when the greater proportion of the c e l l s in the testis were mature spermatids and sperm (stage 4) protamine synthesis had also largely ceased. Incorporation of [1''C]-arginine into proteins occurred in a l l stages of salmonoid testes, with the highest incorporation occurring during stage 2 and 3 and then decreasing drastically in stage 4 testes. It can be noted from table 2 that the major class of proteins synthesized by testis c e l l s at a l l stages are the acid insoluble proteins representing greater than 60 % of the proteins synthesized. Presumably, these acid insoluble proteins consist mainly of essential synthetic and catabolic enzymes, glycolytic and c i t r i c acid cycle enzymes, proteins of the membranes, mitotic and meiotic apparatus, proteins of the sperm t a i l , non-basic chromosomal proteins and enzymes i n -volved in egg penetration. In stage 4 testes, an increase in the amount of acid insoluble protein synthesized to 95.0 % of the total product would be coincident with the maturation of the sperm, i.e., the synthesis of t a i l protein and enzymes involved in egg penetration, and the decrease in the synthesis of protamine. The data in table 3 show that the total protein content per testes increases with maturation. However, the protein content of testis tissue (mg/g) f i r s t decreases from a value of 2 09 mg/g (stage 1) to a value of 128 mg/g (stage 3) and then increases to a value of 261 mg/g (stage 4). The decrease in protein content (mg/g) is l i k e l y due to the drastic reduction I 100. T a b l e 3. Percent a r g i n i n e i n t o t a l p r o t e i n o f the v a r i o u s stages o f O. tschawytscha t e s t e s . Stage Average • T e s t i s Weight g P r o t e i n Content mg/g T o t a l P r o t e i n g / t e s t i s P e r c e n t A r g i n i n e % 1 24 199 4.8 7.54 218 5.2 8.07 2 137 167 22.9 8.23 160 21.9 8 .05 3 708 128 90.6 20.50 127 90.0 19.00 4 440 284 125.0 28.00 238 105.0 32.40 Average t e s t i s weight f o r 75 cm f i s h . V a l u e s taken from f i g u r e 1. The stage 1 sample c o n s i s t e d o f p i e c e s o f t i s s u e from ten stage 1 t e s t e s . The weight of the t e s t e s ranged from 15.4 to 27.8 g ( s t a n d a r d i z e d f o r 75 cm f i s h ) ; the average weight being 2.12 g. Stage 2 and stage 3 samples were taken from one f r o z e n t e s t i s each; 184 g and 669 g, r e s p e c t i v e l y ( s t a n d a r d i z e d f o r 75 cm f i s h ) . The stage 4 sample c o n s i s t e d o f p i e c e s of t i s s u e from two stage 4 t e s t e s , 510 g and 480 g ( s t a n d a r d i z e d f o r 75 cm f i s h ) . P r o t e i n c o n t e n t was determined by the b i r u e t method (253) on d u p l i c a t e samples o f t e s t i s p r o t e i n i s o l a t e d from each stage o f t e s t e s as d e s c r i b e d i n M a t e r i a l s and Methods. D u p l i c a t e samples o f t e s t i s p r o t e i n were h y d r o l y z e d and then a c e t y l a t e d . The a r g i n i n e c o n t e n t of these samples were determined by the Sakaguchi r e a c t i o n (256) . 101. in the amount of interlobular connective tissue separating the cysts of c e l l s and the quantitative reduction of the cytoplasmic protein fraction that occurs during testis development. The increase in protein content (mg/g) during the last stage of testis development, a period when protein synthesis has decreased drastically, i s l i k e l y due to the condensation of the nuclei df spermatids during their transformation into spermatozoa. To test the method for estimating arginine content of proteins, bovine serum albumin (Armour Pharmaceutical Co.) was hydrolyzed, acetylated, and i t s arginine content determined by the Sakaguchi reaction (256). Bovine serum albumin was found to be 5.5 % arginine which corresponds well to the value stated by Neurath (311) of 5.9 % arginine. The data in table 3 show a dramatic increase in the arginine content of the total testis protein during salmon testis mat-uration; from approximately 8 % in stage 1 and 2, to 19.8 % in stage 3, to a high value of 30.2 % in stage 4. This data complements the observation (24) that the arginine content of isolated trout nuclei increases dramatically during trout testis maturation. This large increase in the arginine content of total testis protein during spermatogenesis i s l i k e l y the result of two combined effects; (i) the extreme qualitative modification of basic nuclear proteins to protamines consisting predominantly of arginine residues and (ii) the extreme quan-ti t a t i v e reduction of other nuclear and cytoplasmic protein fractions. 102. The maturing salmon testis system appears to be a good system in which to study developmental changes in tRNA because, at the various stages of spermatogenesis, portions of the pro-tein synthesizing system of testis c e l l s are committed to the synthesis of different specialized proteins. During the i n i t i a l phase of spermatogenesis, replication of the c e l l ' s DNA and concomitant synthesis of i t s chromosomal proteins seem of paramount importance. Thus, a large portion of protein syn-thesis in stage 1 and stage 2 testes (29 % and 36 %, respec-tively, table 2) i s directed towards the synthesis of basic nuclear proteins. During maturation of the spermatid, the DNA of the testis c e l l i s transformed into a tightly packed, metabolically inactive form by the replacement of histones by protamines. In stage 3 testis, therefore, where this transformation i s occurring, 16 percent of the c e l l ' s protein synthesis (table 2) and 73 % of i t s incorporated arginine (table 1) are committed to protamine synthesis. The synthesis of the two types of basic nuclear protein d i f f e r dramatically in stage 1, stage 2, and stage 3; nuclear basic protein syn-thesis consisting mainly of histones in stage 1, an equal amount of histone and protamine in stage 2, and three times the amount of protamine as histone in stage 3. During the f i n a l stage of maturation, protein synthesis in testis c e l l s i s drastically decreased, basic nuclear protein synthesis has almost ceased and 95 percent of the c e l l ' s remaining protein synthesis i s directed towards the synthesis of acid insoluble proteins (table 2), l i k e l y to the synthesis of the microtubular protein of sperm t a i l s and the enzymes involved in egg penetration. II. Transfer RNA Extraction The testis tissue of 0. tschawytscha has a high content of DNA when compared to other tissue. From table 5 i t can be seen that stage 4 testes have a DNA content of 68 mg/g whereas salmon l i v e r has a DNA content of 2.7 mg/g. Therefore, a method of extraction giving good yields of active tRNA with l i t t l e DNA contamination was essential for the study of tRNA in salmon testes. The modified Brunngraber1s extraction procedure (see Materials and Methods) which involves the direct homogeniza-tion of tissue in a buffer-phenol solution of pH 7.5 gave con-siderable contamination of tRNA preparations with DNA. From table 4 i t can be seen that the salmon li v e r tRNA was contam-inated with 7.5 % DNA and salmon testis tRNA was contaminated with 15.2 % DNA when prepared by this method. However, when homogenization of the tissue was performed in a buffer-phenol solution of pH 4.7, DNA contamination of tRNA was drastically decreased, and in the case of salmon liver tRNA, was less than 1 %. For this reason, a l l tRNA preparations studied in this research used this pH 4.7 extraction method. Since the quantitative yield of tRNA from various stages of salmon testes was to be determined, f a i r l y pure samples of Table 4. DNA Content of tRNA Preparations. Method of tRNA Sample Percent DNA (%) Extraction phenol - pH 7.5 salmon l i v e r 7.5 phenol - pH 4.7 salmon l i v e r < 1.0 phenol - pH 7.5 salmon testes 15.2 Transfer RNA was either extracted from tissue using homogenizing buffer-phenol pH 7.5 or using homogenizing buffer-phenol pH 4.7 by the procedure described in Materials and Methods. The DNA content of the tRNA fraction from the DEAE-cellulose column was determined by the diphenyl-amine method (312). 105. tRNA were required. However, preparations of tRNA can be con-taminated by several other macromolecules besides DNA. For example, during the extraction procedure, high molecular weight RNAs (ribosomal RNA and large messenger RNA) were removed from tRNA preparations by precipitation with 2 M NaCl. Then, by DEAE-cellulose chromatography (Figure 5) the tRNA sample was further purified. Nucleoside triphosphates, phenol and glycogen were a l l eluted from the column before 0.5 M NaCl. The tRNA was eluted from the column between 0.5 and 0.8 M NaCl. Any ribosomal RNA s t i l l contained in the loading solution was bound to the DEAE-cellulose and not eluted from the column. In order to compare yields of tRNA from various stages of salmon testes, the yield of tRNA from one stage of salmon testes must be reproducible. In an experiment in which tRNA was extracted from two samples of salmon testes at the same stage of maturation, the amount of tRNA (mg/g testes) extracted from the two samples was found to d i f f e r by less than 5 percent. This result indicates the reproducibility of the tRNA extraction procedure described in Materials and Methods. III. Changes in Nucleic Acids During Spermatogenesis Salmon testis i s a tissue rich in DNA. The DNA content of immature salmon testes (14.2 mg/g, table 5) i s approximately five-fold greater than that of salmon li v e r (2.7 mg/g, table 5), reflecting the smaller size and high tissue concentration of germ c e l l s . The DNA concentration of testis tissue i s observed Figure 5. DEAE-cellulose chromatography of stage 3 testis tRNA. Transfer RNA was extracted from a stage 3 testis as des-cribed in Materials and Methods. The dialyzate containing 1187 A26o units of ultraviolet-absorbing material was applied to a column (2.5 cm x 25 cm) of DEAE-cellulose (chloride form) and then eluted with a 3 1 gradient running from 0 to 1.5 M NaCl in 0.01 M Tris HCl pH 8.0 at rate of 3 ml/min. The 18 ml fractions collected were monitored at 260 nm. The tRNA fraction i s designated by arrows. The earlier eluting peaks are nucleotide contaminants. 106a A b s o r b a n c e p o 2 6 0 n m b b b U l b b J 3 Table 5. DNA and RNA values of 0., tschawytscha testes and liver during the various stages of sexual maturation. Stage of Testes Average Tissue Weight DNA RNA Total DNA Total RNA g mg/gb mg/gb g/tissue g/tissue 1 24 14.2 9.25 0.33 0.22 2 137 23.1 6.55 3.16 0.90 3 708 38.8 4.60 27.42 3.26 4 440 67.7 1.40 29.80 0.62 Salmon liver 81 2.7 7.60 0.22 0.62 a Average testis and liv e r weight for 75 cm f i s h . Expressed as mg/g of tissue (wet weight). DNA and RNA content of testis and liver tissue was determined by procedures des-cribed in Materials and Methods. These data are the average of two values obtained from the tissue of two f i s h at the appropriate stage. to increase further during sexual maturation, from 14.2 mg/g to 67.7 mg/g (table 5). DNA analyses also indicate that DNA synthesis has almost completely ceased in stage 3 testes. This is expected because stage 3 testes are composed mostly of sperm-atids, c e l l s synthesizing very l i t t l e DNA (26). The RNA content of 0. tschawytscha testes decreases from 9.25 mg/g to 1.4 0 mg/g (table 5) as the testes mature. Other investigators (24,26,32) have noted a similar decrease in the RNA content during testis maturation of other salmonids. These results indicate that the RNA i s lost from the whole tissue and not just from the maturing sperm c e l l . RNA loss during sexual maturation i s a very extensive process involving complete breakdown in, or expulsion from, the testes. Ling and Dixon (29) have observed, as rainbow trout testes mature, a continuous decrease in total ribosomes per gram testes, A study investigating the tRNA content of maturing 0. tschawytscha testes (table 6) has established that, similarly, a decrease in tRNA occurs with testis maturation. A continuous decrease in total tRNA per gram testes occurs from stage 1 testes (425 Vg/q) to stage 3 testes (246 yg/g). At stage 4 there i s even a sharper decrease in tRNA content down to a value of 66 yg/g. Salmon milt which contains l i t t l e or no RNA (24,33-35) contains a very small amount of tRNA (2 yg/g). This tRNA may not be cytoplasmic, because mature sperm have l i t t l e , i f any, cytoplasm. However, since mature sperm of teleosts contain mitochondria (7,314), salmon milt tRNA may originate from these organelles. 1 0 9 . Table 6. RNA values of 0. tschawytscha testes and liv e r during the various stages of sexual maturation. Stage Total RNA tRNA Extracted tRNA Corrected tRNA Percent of Total RNA mg/ga mg/ga mg/ga % 1 9 . 2 5 0 . 4 5 2 0 . 9 0 4 9 . 8 2 6 . 5 5 0 . 3 0 7 0 . 6 1 4 9 . 4 3 4 . 6 0 0 . 2 4 6 0 . 4 9 2 1 0 . 6 4 1 . 4 0 0 . 0 6 6 0 . 1 3 2 9 . 4 Salmon Milt - 0 . 0 0 2 - -Salmon Liver 7 . 6 0 0 . 3 6 6 0 . 7 3 2 9 . 6 Expressed as mg/g of tissue (wet weight). RNA content of testis and li v e r tissue taken from table 5. Salmon li v e r tRNA was extracted from the liv e r of a stage 1 f i s h . Stage 1 testis tRNA was extracted from ten stage 1 testes. The weight of these testes ranged from 15.4 to 27.8 g (standard-ized for 75 cm fish); the average being 21.2 g. Stage 2 and stage 4 tRNA were extracted from one frozen testis each, 184 g and 481 g, respectively. Stage 3 tRNA was extracted from one frozen tes t i s , 6 6 9 g (standardized for 75 cm fis h ) ; the same testis used for the [ 1 **C]-arginine incorporation study (table 1). Salmon milt tRNA was extracted from 200 g of frozen milt collected from chinook salmon at the Green River Hatchery. The yield of tRNA was determined from A 2 6o value of the tRNA peak from the DEAE-cellulose column (see Materials and Methods). Because 2 M NaCl has been shown recently to precipi-tate 50 % of the total tRNA with the high molecular weight RNA (313) yields of tRNA were increased by a factor of 2 to correct for loss of tRNA at this step. Furthermore, the recent results of Premkumar and Bhargava (43), make the mitochondrial origin of salmon milt tRNA even more credible. These researchers found that in mature, fresh bovine sperm most, i f not a l l , transcription and translation i s mito-chondrial. They also found that bovine spermatozoa synthesize a RNA species which on methylated albumin kieselguhr columns, behaves li k e bacterial tRNA's, rather than mammalian cytoplasmic tRNA, a property characteristic of mitochondrial tRNA. However, because salmon sperm contains such a small quantity of tRNA, i t did not seem practical to study the properties or establish the origin of this tRNA. The content of liv e r tRNA was deter-mined at only one stage of maturation (stage 1); the RNA content of l i v e r , unlike that of testes, being independent of the sexual maturation of the f i s h . The tRNA fraction from testes at the four stages of develop ment, like that of l i v e r , make up approximately 10 percent of the total RNA content (table 6). The presence of tRNA in the same proportions at a l l stages of testis development indicates that the RNA decrease observed during 0. tschawytscha testis maturation must be a specific and well regulated process. This drastic decrease in RNA content during testis matura-tion of salmonids is probably the result of two combined effects a decreased rate of RNA synthesis during spermatogenesis and the active extrusion of RNA from the maturing sperm c e l l . A progressive decrease in RNA synthesis has been noted in the maturing testis c e l l s of mammals (36) and insects (37) and no RNA synthesis was found in their maturing spermatids. The meiotic nucleolus of l i l y microsporocytes (male gametocytes) appears to reduce both the rate of rRNA transcription and ribosomal maturation (116). The decline in RNA synthesis during spermatogenesis could occur by an inhibition of template activity and/or by the decrease in the levels and a c t i v i t i e s of the multiple RNA polymerases. Preliminary studies (315) i n d i -cate that during the maturation of rainbow trout (Salmo gairdnerii) testes there i s a progressive and an approximately proportional decrease in both nucleoplasmic and nucleolar RNA polymerase a c t i v i t i e s . Experiments of Roeder (316) indicate the absence of detectable RNA polymerase activity in the nuclei of mature sea urchin sperm and nuclei of mature trout sperm. It seems that the amount of tRNA and rRNA present at a particular stage of development may not reflect the rate of protein synthesis. For example, stage 1 testes synthesizes less protein than stage 2 and 3 (table 1) and contains more ribo-somes and tRNA per gram testes. However, the drastic decrease in protein synthesis in stage 4 testes may result from the sharp decrease in RNA content, either due to a lack of mRNA or to limiting quantities of ribosomes and tRNA. IV. Properties of Arginyl-tRNA Synthetase Preparations Because the arginine content of total testis protein i n -creases by 370 percent during salmon testis maturation and specifically because this amino acid i s a major component of protamine, changes in arginine tRNA of maturing salmon testes were investigated in d e t a i l . This required an investigation of certain physical and kinetic properties of the arginyl-tRNA synthetase preparations, used to acylate tRNA A r g. These prop-erties of arginyl-tRNA synthetase preparations w i l l be described in the following pages. (a) Heat l a b i l i t y of salmon li v e r arginyl-tRNA synthetase  preparation Most tRNA acceptor assays are performed at either room temperature or at the optimum temperature of 37°. However, salmon enzymes may be heat-labile at these temperatures because salmon are cold-blooded animals l i v i n g in fresh or salt water habitats with temperatures less than 10°. Figure 6 shows the results bf the effect of temperature on the extent of arginine acceptance of salmon testis tRNA in 15 min when using a salmon l i v e r aminoacyl-tRNA synthetase preparation. Optimum aminoacylation occurred at 23° and this was the temperature used for a l l subsequent aminoacylation i n -cubations. It should be noted that considerable aminoacylation occurred even at 4°. The arginyl-tRNA synthetase i s probably heat-labile because l i t t l e aminoacylation occurred at 37°. This heat-labile synthetase i s unlike the phenylalanyl-tRNA synthetase of trout l i v e r studied by Rosen et a l . (265) which aminoacylated rat l i v e r tRNA with phenylalanine to a greater 113. Figure 6. The effect of temperature on the extent of arginine acceptance of salmon testis tRNA in 15 min when using a salmon li v e r aminoacyl-tRNA synthetase prep-aration . Arginine acceptor activity was measured by the procedure described in Materials and Methods (p. 71). Incubation mixtures (0.2 ml) contained, in addition to components mentioned in this section, 0.171 A 2 6 o units stage 4 salmon testis tRNA, 1.7 nanomoles arginine (0.2 yCi [ 1^C]-arginine) and 50 ymoles NaCl. Mixtures were incubated at the various temperatures (4°, 14°, 23°, and 37°) for 15 min and then 50 yl samples withdrawn. 114 . extent in 15 min at 37° than 23°. However, whether heat-l a b i l i t y i s a characteristic of a few or a l l salmon l i v e r aminoacyl-tRNA synthetases has not been determined. (b) Stability of salmon l i v e r arginyl-tRNA synthetase prep-arations From table 7 i t can be seen that crude salmon li v e r arginyl-tRNA synthetase preparations in 40 % (v/v) glycerol retain after prolonged storage (41 or 104 days) at -20° sufficient enzyme activi t y to f u l l y charge the tRNA A r g present in the incubation mixture. (c) Kinetic properties of salmon li v e r arginyl-tRNA synthetase The effect of varying concentrations of arginine on the formation of arginyl-tRNA by salmon l i v e r synthetase i s shown in figure 7. The Km value for arginine determined from the Lineweaver-Burk plot of this data (figure 8) i s 0.19 yM. This i s a much lower Km value for arginine than that of rat liver arginyl-tRNA synthetase; the Km value for arginine being 1.25 yM (317). The approximate ten-fold difference in a f f i n i t y for arginine between the two l i v e r arginyl-tRNA synthetase prep-arations i s not unprecedented because synthetases d i f f e r widely in their Km value for amino acids (318) . However, i t should also be noted that a direct comparison between the Km values for arginine of these two l i v e r arginyl-tRNA synthetases i s not applicable because the Km values were determined under different assay conditions (temperature and Mg++/ATP ra t i o ) . Table 7. Stability of salmon li v e r arginyl-tRNA synthetase preparations. Enzyme Amount of tRNA per Freshly Prepared 41 Day Old Prep 0.2 ml mix Enzyme Enzyme A 2 6 0 units pmoles arginine accepted/ 0.2 ml mix 0.171 18.44 20.21 0.342 34.20 34.96 Enzyme Amount of tRNA per Freshly Prepared 104 Day Old Prep 0.2 ml mix Enzyme Enzyme B A 2 6 o units pmoles arginine accepted/ 0.2 ml mix 0.066 7.84 7.12 0.166 17.80 17.28 Arginine acceptor activity was measured by the procedure described in Materials and Methods (p. 71). A l l incubation mixes contained 2 nanomoles arginine (0.2 yCi [ 1 **C] -arginine) . The amount of stage 4 salmon testis tRNA in each incubation mix i s stated in the table. Mixtures were incubated for 4 0 min with enzyme A and 3 0 min with enzyme B before samples were withdrawn. Figure 7. The effect of arginine concentration upon the form-ation of arginyl-tRNA by a salmon liver arginyl-tRNA synthetase preparation. < Z 10 c < o E a • • i i i i i i i i i i O l 2 3 4 5 6 7 8 9 10 A r g i n i n e CpM) Arginine acceptor activity was measured by the procedure described in Materials and Methods (p. 71). Incubation mixtures contained 0.207 A2eo units (1.39 yM) stage 4 testis tRNA and varying concentrations of arginine. Samples were withdrawn after 60 min incubation. Counts were corrected for values obtained for the controls (at each arginine concentration) to which no RNA was added. igure 8. The Lineweaver-Burk plot of a salmon li v e r arginyl-tRNA synthetase preparation for determination of the Km value for arginine. .20 The effect of salmon testis tRNA concentration upon the formation of arginyl-tRNA by salmon li v e r arginyl-tRNA syn-thetase is shown in figure 9 . From this figure i t i s apparent that the extent of acceptance of arginine, in the presence of excess enzyme, is proportional to the amount of tRNA added within limiting concentrations. The Km value for salmon testis (stage 4) tRNA determined by the Lineweaver-Burk plot of this data (figure 10 ) i s 0 . 7 6 yM. As expected, this Km value for tRNA i s within the normal range of 1 0 ~ 7 M found for a l l amino-acyl-tRNA synthetases ( 3 1 8 ) . (d) Assaying for true arginyl-tRNA formation Salmon l i v e r arginyl-tRNA synthetase preparations could well contain arginyl-tRNA protein transferase, an enzyme which specifically catalyzes the transfer of arginine from arginyl-tRNA into peptide linkage with the amino-terminal residue of certain protein acceptors (273 - 2 7 7 ) . If during assay pro-cedures, arginine should be linked to proteins as well as to tRNAAr^, cold TCA acid insoluble incorporation would give a value higher than that for true arginyl-tRNA formation. A true value for aminoacyl-tRNA formation i s obtained by deter-mining the difference between cold and hot TCA acid insoluble incorporation ( 2 7 4 ) . The true value for arginyl-tRNA formation by a salmon liv e r arginyl-tRNA synthetase preparation (hot TCA labile fraction) was found to be 84 - 8 6 percent of the total cold 119. Figure 9. The effect of salmon testis tRNA concentration upon the formation of arginyl-tRNA by a salmon li v e r arginyl-tRNA preparation. Arginine acceptor activity was measured by the procedure described in Materials and Methods (p. 71 ). Incubation mixtures contained 2 nanomoles arginine (0.2 yC [1''C]-arginine and varying concentrations of stage 4 testis tRNA (0.007 - 0.214 A 2 6 o units). The molar concentration of tRNA in the incubation mixes was calculated knowing that 1 mg of tRNA in 1 ml of d i s t i l l e d water has an absorbance of 24 at 260 nm and that the molecular weight of tRNA i s approx-imately 26,500 (319). Samples were withdrawn after 60 min. pmoles ' A r g i n y l - t R N A Fo rmed /60 m i n 120. TCA acid insoluble incorporation (table 8 ) . Because the arginine transferase reaction i s highly dependent on added tRNA ( 2 7 4 ) , more arginine was linked to protein (expressed as hot TCA acid insoluble incorporation, table 8) as the tRNA concentration of the incubation mixes was increased. However, the porportion of the total arginine incorporated which was linked to protein remained approximately the same (13 - 16 per-cent) at different tRNA concentrations. It i s l i k e l y that minimal arginyl-tRNA protein transferase activity i s observed in the assay mixtures (pH 7.5) because the transfer reaction i s dim-inished at pH 7.4 by almost 70 percent from the value at the pH optimum (9.0 - 9.8) ( 2 7 5 ) . When a true value for the tRNA A r g content of samples was needed in this research, the arginine incorporation into the hot TCA labi l e fraction was determined. However, incorporation of arginine into cold TCA acid insoluble fraction was used to measure the tRNA A r g content of column fractions. Determination of the cold TCA acid insoluble fraction alone i s more convenient. Furthermore, evidence (277) indicates that v i r t u a l l y a l l the arginyl-tRNA rather than just an unique molecular species, participates in the arginyl-tRNA protein transferase reaction. Therefore, the content value for a l l isoaccepting species of tRNA A r g in column fractions measured by cold TCA method w i l l be approximately 15 percent higher than the true value measured by hot TCA labile incorporation. Table 8. Assay of true arginyl-tRNA formation tRNA 5 % TCA (0°) a 5 % TCA (90°) b (0° Value-90° Value) C A 2 6 o units/mix pmoles Arg pmoles Arg pmoles Arg Percent of Total True Arginyl-tRNA Formation 0.000 0.069 0.171 0.342 1.03 8.50 19.5 37.0 0 . 6 5 1 . 4 0 3 . 0 3 5 . 0 3 0 . 3 8 7 . 1 0 1 6 . 4 3 2 . 0 83.6 84.4 86.4 Indicates the amount of arginine incorporated onto the termini of proteins plus the amount of arginyl-tRNA formed per 0.2 ml mix. Indicates the amount of arginine incorporated onto the termini of proteins per 0.2 ml mix. c Indicates the true amount of arginyl-tRNA formed per 0.2 ml mix. True arginyl-tRNA formation was measured by the procedure described in Materials and Methods (p. 73). A l l incubation mixes (0.2 ml) contained 2 nanomoles arginine (0.2 yCi [ 1^C]-arginine). The amount of stage 4 salmon testis tRNA contained in each incubation mixture is described in the table. Samples of 100 yl were withdrawn from duplicate incubation mixtures after 60 min. Whereas one sample was treated with 5 % (w/v) TCA at 0°, the other sample was treated with 5 % (w/v) TCA at 90°. (e) The effect of ethanol and various NaCl concentrations  on arginyl-tRNA formation In the course of assaying BD-cellulose column fractions for arginine acceptor activity, i t was noted that the arginine acceptor activity decreased i f the aliquots of the column frac-tions introduce significant amounts of salt into the assay mixture. For example, a 50 y l aliquot of a fraction contain-ing approximately 38.5 ymoles NaCl formed 0.456 nanomoles of arginyl-tRNA/ml, whereas, a 100 yl aliquot containing approx-imately 75 ymoles NaCl formed 0.3 99 nanomoles arginyl-tRNA/ml. A further investigation on the effect of NaCl solutions on the a b i l i t y of a fixed amount of tRNA to accept arginine i s shown in figure 11. The aminoacylation values in figure 11 represent the amount of arginine accepted at the plateau, i.e., at the completion of aminoacylation. The addition of 25 ymoles NaCl slightly increased the amount of arginine accepted by the tRNA. Maximal arginyl-tRNA formation occurred with the addition of 25 ymoles to 50 ymoles NaCl. The amount of arginine accepted by the tRNA decreased with the addition of 50 ymoles to 100 ymoles NaCl. From figure 11, one can see that i f 75 ymoles, rather than 38.5 ymoles of NaCl, are added to the amino-acylation reaction, arginyl-tRNA formation i s decreased by 11 percent. This i s very similar to the 13 percent decrease in arginyl-tRNA formation noted when a 100 y l aliquot, rather than a 50 y l aliuqot from the BD-cellulose column fraction, was assayed. Because a decrease in arginyl-tRNA formation is ob-served at NaCl concentrations greater than 50 ymoles/0.2 ml, F i g u r e 11. The e f f e c t of e t h a n o l and v a r i o u s NaCl concen-t r a t i o n s on a r g i n y l - t R N A f o r m a t i o n . 24 • i 1 1 1 1 1 1 1 1 i— 0 2 0 4 0 6 0 8 0 1 0 0 umoles N a C l Arginyl-tRNA f o r m a t i o n was measured a c c o r d i n g to the p r o -cedure d e s c r i b e d i n M a t e r i a l s and Methods (p. 71). A l l i n c u -b a t i o n m i x t u r e s (0.2 ml) c o n t a i n e d 20 umoles sodium c o c o d y l a t e (pH 7.5), 0.5 ymoles sodium ATP, 2 ymoles M g C l 2 , 80 nanomoles sodium EDTA, 2.0 nanomoles a r g i n i n e (0.2 y C i [ 1 **C] - a r g i n i n e ) and 0.171 A 2 6 o u n i t s of stage 4 salmon t e s t i s tRNA. D i f f e r i n g amounts of NaCl (0 - 100 ymoles) were added to the. i n c u b a t i o n mixes. Ten m i c r o l i t r e s o f 95 % e t h a n o l was added to the i n -c u b a t i o n mix ( O ) . a l l BD-cellulose column fractions were diluted before assay-ing for arginine acceptor activity. Since the arginine acceptor activity of BD-cellulose column fractions eluted with 1 M NaCl and 10 % ethanol were also determined during this research, the effect of ethanol on arginyl-tRNA formation was investigated (figure 11). The amount of arginine accepted by the tRNA in a mix containing 100 ymoles NaCl decreased 18 percent in the presence of 4.8 % ethanol. Because ethanol was found to inhibit arginyl-tRNA formation, ethanol was evaporated under vacuum at 3 0° from the column fraction before the column fractions were assayed. (f) Effect of Sephadex G-25 chromatography on the activity of salmon l i v e r and salmon testis arginyl-tRNA synthetase  preparations From figure 12 i t can be seen that a crude salmon li v e r arginyl-tRNA synthetase preparation gives maximal charging at a l l enzyme concentrations tested. A crude salmon testis arginyl-tRNA synthetase preparation shows less arginyl-tRNA formation at a l l enzyme concentrations when compared to the crude salmon li v e r arginyl-tRNA synthetase preparation (figure 12). Also, as the concentration of salmon testis arginyl-tRNA synthetase in the incubation mixes increases, a decrease in arginyl-tRNA formation i s observed. In contrast, a salmon testis arginyl-tRNA synthetase preparation, after Sephadex G-25 chromatography, acylated an amount of tRNA A r g similar to that acylated by the salmon l i v e r arginyl-tRNA synthetase (figure Figure 12. The effect of Sephadex G-25 chromatography on arg-inine acceptor activity of aminoacyl-tRNA synthetase preparations. x £ o <D (J O CD C ' c O) o 0 £ Uni ts e n z y m e ( m g t issue) Arginine acceptor acti v i t y was measured by the procedure described in Materials and Methods (p. 71). The incubation mix (0.2 ml) contained, in addition to components stated in this section, 0.166 A 2so units stage 4 salmon testis tRNA and 2 nanomoles arginine (0.2 yCi [ 1 **C]-arginine) . Samples of 50 yl were removed after 3 0 min. A l l arginyl-tRNA synthetases were prepared from tissue of a stage 2 salmon by the procedure described in Materials and Methods (p. 68) except that salmon testis arginyl-tRNA synthe-tase ( • ) was chromatographed on a Sephadex G-25 column whereas salmon testis ( • — ) and salmon liver ( — • — ) arginyl-tRNA synthetases were not chromatographed on Sephadex G-25 columns. Units of enzyme expressed as mg tissue, i.e., the amount of tissue (wet weight) from which 50 y l of enzyme was extracted. 12), and showed l i t t l e change in the amount of arginyl-tRNA formed with increased enzyme concentration. The difference in a c t i v i t i e s of the crude and the part-i a l l y purified salmon testis arginyl-tRNA synthetase prepara-tions could be explained i f the crude salmon testis arginyl-tRNA synthetase preparation contained sufficient arginine to decrease the specific activity of the substrate. Sephadex G-25 chromatography would remove amino acids and hence the salmon testis arginyl-tRNA synthetase purified by Sephadex G-25 chromatography would show l i t t l e change in the amount of arginyl-tRNA formed with increased enzyme concentration. Unlike testis arginyl-tRNA synthetase preparations, the li v e r arginyl-tRNA synthetase preparation apparently does not contain a large amount of arginine because there was l i t t l e change in the amount of arginyl-tRNA formed with increased enzyme concentration. It would be interesting to determine the arginine content of the cytoplasmic enzyme preparation of stage 2 testes, since testes at this stage of maturation are using large amounts of arginine for the synthesis of protamine (table 1). V. Relationship of tRNA A r g and tRNA L y s to basic nuclear  protein synthesis in salmon testes Studies discussed in the introduction (226-230, 232-238, 24 0-24 5) have attempted to correlate quantitative changes in the amounts of specific tRNA's with changes in the amino acid composition of proteins being synthesized by various tissue. 128 . In a l l these systems, the types of proteins being synthesized changed markedly. It appears that gene amplification i s not a factor in such systems (72-74). A similar situation occurs during salmon testis maturation because testis proteins have a markedly different arginine content at the various stages (approximately 8 % at stage 1 and 30 % at stage 4, table 3). The increase in the arginine content of testis protein during spermatogenesis i s coincident with the replacement of histones by protamines, which contain approximately 67 % arginine. If, therefore, the intracellular tRNA content i s related to the amino acid composition of proteins being synthesized, one would expect tRNA extracted from maturing salmon testes to be enriched with arginine-accepting species. This po s s i b i l i t y was examined by determining the capacity of tRNA preparations from salmon testes at various stages of maturation to accept arginine, lysine, serine, proline, glycine, and aspartic acid. The results were compared with the amino acid acceptance of a tRNA preparation from salmon l i v e r . The l i v e r was chosen for this comparison because the variety of proteins i t synthesizes for both internal use and for extra-cellular transport would suggest i t is relatively nonspecial-ized in i t s u t i l i z a t i o n of amino acids and therefore in i t s tRNA content. In order to quantitate the relative amount of these specific tRNA's within salmon li v e r and salmon testis tRNA preparations, aminoacyl-tRNA formation was measured at a limiting concentration of tRNA (0.05 to 0.20 A 2 6o units per 0.2 ml mix) in the presence of excess ATP, amino acid and enzyme. The concentration of enzyme and the incubation time needed to assure a linear response to added tRNA was determined in pre-^ liminary testes with each amino acid. A testis aminoacyl-tRNA synthetase preparation from a stage 2 salmon, purified by Sephadex G-25 chromatography to remove endogenous amino acids, was used to measure the amino acid acceptance of these tRNA preparations. Although arginyl- and lysyl-tRNA synthetase a c t i v i t i e s remained stable after storage for several months, experiments indicated that seryl-, glycyl-, prolyl-, and aspartyl-tRNA synthetase a c t i v i t i e s were decreased with storage. Therefore, in order that enzyme s t a b i l i t i e s would not affect results, the assays for one amino acid were a l l performed on the same day. Since duplicate samples were withdrawn from each incubation mixture and amino acid acceptance was deter-^ mined at two tRNA concentrations, four values were obtained for the amino acid acceptance of each tRNA preparation, The average of the four amino acid acceptance values for each tRNA preparation i s expressed in table 9. In a l l cases, except serine at stage 4B, a standard error of less than 10 % i s seen. Besides expressing results in table 9 in pmole/A26o unit, amino acid acceptance a c t i v i t i e s are expressed in pmoles/ pmoles glycine accepted. This latter reference i s used because different amounts of contaminating ultraviolet-absorbing material may be contained in the tRNA preparations. Table 9. The amino acid acceptance ac t i v i t i e s of tRNA preparations from salmon liver and various stages of salmon testes. Salmon li v e r tRNA was extracted from the liver of a stage 1 f i s h . Stage 1 testis tRNA was extracted from ten stage 1 testes. The weight of the testes ranged from 15.4 to 27.8 g; the average being 21.2 g. Stage 1A testis tRNA was extracted from two testes (60 g and 62 g) obtained from salmon 8 weeks into testis maturation (beginning the logarithmic phase of testis growth). Stage 2 tRNA was extracted from one frozen testes, 184 g. Stage 3 tRNA was extracted from one frozen testis, 669 g; the same testis used for the t 1^C]-arginine incorporation study (table 1). Stage 4A and 4B tRNA were each extracted from one frozen testis, 481 g and 545 g, respectively. A l l testis weights were standardized for 75 cm f i s h . Stage 4B was the same testis used in [ 1 "*C]-arginine incorporation study (table 1). This testis was "riper" in appearance and had smaller tRNA content (0.037 mg/g) than the stage 4A testis (0.066 mg/g). Assay systems (0.2 ml) contained approximately 0.07 A 2 6 0 units or 0.175 A 2 6 0 units of the specified tRNA. The remaining components of the assay systems were previously described in Materials and Methods. After the incubation period, duplicate 50 yl samples were removed and in the case of a l l amino acids except arginine processed as described in Materials and Methods (p. 71 ). In the case of arginine, duplicates were processes as for the determination of true arginyl-tRNA formation (Materials and Methods p. 73 ) . o co Table 9 Source of tRNA Amino Acid Acceptance (pmoles/A2 6 0 unit) Arg Lys Ser Asp Pro Gly Salmon liver 67 .5 ± 2 .3 a 30 .3 + 0.4 85.0 ± 3.0 67.5 + 3.0 37.4 + 0. 0 38 .3 ± 0. 8 Stage 1 testes 80 .9 ± 3 .9 31 .9 ± 0.9 59.9 ± 0.1 24.4 ± 0.3 31.1 ± 0. 3 25 .7 ± 1. 0 Stage 1A testes 91 .7 + 3 .1 39 .2 ± 0.3 65.0 + 1.1 33.3 ± 1.6 18.3 ± 0. 1 23 .9 ± 1. 1 Stage 2 testes 93 .1 ± 1 .4 41 .4 ± 2.6 67.4 + 1.5 42.0 ± 0.3 20.2 + 0. 6 22 .0 ± 0. 4 Stage 3 testes 131 .7 i 5 .4 29 .1 ± 1.0 72.5 ± 1.5 42.0 ± 0.3 25.9 + 1. 3 26 .4 + 1. 2 Stage 4A testes 132 .5 ± 3 .1 39 .5 ± 0.5 74.2 + 0.2 44.3 ± 1.0 18.1 + 0. 2 25 .5 ± 1. 7 Stage 4B testes 94 .8 ± 1 .5 28 .6 ± 1.1 66.4 ± 8.4 40.0 ± 1.5 25.4 + 1. 0 20 .0 ± 1. 2 Amino Acid Acceptance (pmoles/pmoles Gly) Salmon liver 1. 76 0.79 2.22 1.76 0.98 1.00 Stage 1 testes 3. 14 1.24 2.33 0.95 1.21 1.00 Stage 1A testes 3. 84 1.64 2.72 1.39 0.76 1.00 Stage 2 testes 4. 24 1.88 3.06 1.91 0.92 1.00 Stage 3 testes 5. 00 1.11 2.85 1.59 0.98 1.00 Stage 4A testes Stage 4B testes 5. 4 . 20") 74 ) 4.97 1.55T V l . 49 1.43) 2. 91") V3.ll 3.31* 1.73-) (1.87 2.0Q> 0.71") (0 1.27> .99 1.00 1.00 Deviation shown i s the standard error The v a l u e s f o r the r e l a t i v e amount of v a r i o u s tRNA's i n the l i v e r and t e s t e s , t o be meaningful, should be the average v a l u e s of tRNA p r e p a r a t i o n s from s e v e r a l d i f f e r e n t t i s s u e samples. Stage 1 t e s t i s tRNA was e x t r a c t e d from 10 stage 1 t e s t e s ; stage 1A t e s t i s tRNA, from two stage 1A t e s t e s . Thus, the r e l a t i v e amounts of v a r i o u s tRNA's i n stage 1 and 1A t e s t i s tRNA p r e p a r a t i o n s are the average of the amounts from these v a r i o u s t e s t e s . However, v a l u e s f o r l i v e r , stage 2 and stage 3 t e s t e s came from o n l y one t i s s u e sample. Stage 4 t e s t i s tRNA was e x t r a c t e d s e p a r a t e l y from two stage 4 t e s t i s samples. The r e l a t i v e amount of a r g i n i n e tRNA (pmoles arginyl-tRNA/pmoles glycyl-tRNA) and l y s i n e tRNA (pmoles lysy1-tRNA/pmoles g l y c y l -tRNA) i n these two stage 4 t e s t i s tRNA p r e p a r a t i o n s (A & B) d i f f e r e d * by o n l y 9.3 % and 8.0 %, r e s p e c t i v e l y ( t a b l e 9). However, r e l a t i v e amounts of a r g i n i n e tRNA i n the v a r i o u s stages of t e s t i s t i s s u e d i f f e r e d * by 30 % (stage 1 and stage 2), 46 % (stage 1 and stage 3) and 16.4 % (stage 2 and stage 3) ( t a b l e 9). S i m i l a r l y , r e l a t i v e amounts of l y s i n e tRNA i n the v a r i o u s stages of t e s t i s t i s s u e d i f f e r e d * by 41 % (stage 1 and stage 2), 53 % (stage 2 and stage 3), and 29 % (stage 3 and stage 4), ( t a b l e 9). The d i f f e r e n c e s i n the r e l a t i v e amounts of a r g i n i n e and l y s i n e tRNA between the v a r i o u s stages of salmon t e s t e s appear, t h e r e f o r e , t o be s i g n i f i c a n t f o r they are much V a r i a t i o n s between stages c a l c u l a t e d by t a k i n g the d i f f e r e n c e between v a l u e s and de t e r m i n i n g i t s percentage of the average between the two v a l u e s . l a r g e r than d i f f e r e n c e s r e l a t i n g t o b i o l o g i c a l v a r i a t i o n between the two stage 4 f i s h . A comparison between the amino a c i d acceptance a c t i v i t i e s (pmole/pmole g l y c i n e accepted) of the stage 1 l i v e r tRNA prep-a r a t i o n and t e s t i s tRNA p r e p a r a t i o n s i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s . The a r g i n i n e acceptance a c t i v i t y of t e s t i s tRNA p r e p a r a t i o n s appears t o be 1.8 t o 3 times g r e a t e r than the l i v e r tRNA p r e p a r a t i o n ; the l y s i n e acceptance a c t i v i t y o f t e s t i s tRNA p r e p a r a t i o n s , 1.4 t o 2.4 times g r e a t e r . The s e r i n e acceptance a c t i v i t y o f t e s t i s tRNA p r e p a r a t i o n s i s onl y s l i g h t l y h i g h e r than the l i v e r tRNA p r e p a r a t i o n . I t s a c t i v i t y v a r i e s from 1.05 t o 1.5 times t h a t of l i v e r tRNA p r e p a r a t i o n . A s p a r t i c a c i d and p r o l i n e acceptance a c t i v i t i e s of t e s t i s tRNA p r e p a r a -t i o n s v a r y ; a t times being s l i g h t l y h i g h e r and a t o t h e r times being s l i g h t l y lower than the l i v e r tRNA p r e p a r a t i o n . More a r g i n i n e and l y s i n e r e l a t i v e t o other amino a c i d s are used f o r p r o t e i n i n t e s t i s t i s s u e than l i v e r t i s s u e because the h i g h e r DNA content of t e s t e s ( t a b l e 5) means a g r e a t e r s y n t h e s i s o f b a s i c n u c l e a r p r o t e i n s , i . e . , p r o t e i n s abundant i n l y s i n e and a r g i n i n e . The enrichment of t e s t i s tRNA popu-l a t i o n s w i t h a r g i n i n e and l y s i n e a c c e p t i n g s p e c i e s ( t a b l e 9) seems r e l a t e d t o t h i s d i s p r o p o r t i o n a t e requirement of a r g i n i n e and l y s i n e f o r b a s i c n u c l e a r p r o t e i n s y n t h e s i s i n t e s t e s . Thus t e s t i s t i s s u e seems to have a tRNA p o p u l a t i o n s p e c i a l i z e d f o r the s y n t h e s i s of b a s i c n u c l e a r p r o t e i n s . Data from t a b l e 9 i n d i c a t e t h a t the content of some i n d i -v i d u a l tRNA's change d u r i n g t e s t i s m a t u r a t i o n . D u r i n g the i n i t i a l phase of t e s t i s m a t u r a t i o n ( l o g a r i t h m i c growth phase --stage 1 t o stage 2) the q u a n t i t y of a r g i n i n e , l y s i n e , a s p a r t i c , and s e r i n e tRNA c o n t a i n e d i n t e s t i s tRNA p r e p a r a t i o n i n c r e a s e d s i g n i f i c a n t l y by 30 %, 41 %, 71 % and 24 %, r e s p e c t i v e l y . Because changes i n the s e r i n e , the a s p a r t i c a c i d and the p r o -l i n e c o n t e n t of p r o t e i n s d u r i n g salmon t e s t i s m a t u r a t i o n have not been s t u d i e d , the amount of t h e i r r e s p e c t i v e tRNA's cannot be c o r r e l a t e d t o any change i n the amino a c i d composition o f t e s t i s p r o t e i n s . However, d u r i n g t h i s phase of t e s t i s matura-t i o n , the s y n t h e s i s of protamine i n c r e a s e s from a low v a l u e of o n l y 0.6 % of t o t a l p r o t e i n s y n t h e s i s i n stage 1 t o a v a l u e of 17.8 % of t o t a l p r o t e i n s y n t h e s i s i n stage 2. The 30 % i n -cre a s e i n a r g i n i n e tRNA con t e n t of the t e s t i s tRNA p r e p a r a t i o n d u r i n g t h i s phase of t e s t i s m a t u r a t i o n c o r r e l a t e s w i t h the requirement f o r a g r e a t e r a v a i l a b i l i t y of a r g i n i n e f o r the s y n t h e s i s o f protamine. The s y n t h e s i s of h i s t o n e s d u r i n g t h i s same p e r i o d of t e s t i s development decreased from a v a l u e of 28.3 % of t o t a l p r o t e i n s y n t h e s i s d u r i n g stage 1 t o 18.1 % of t o t a l p r o t e i n s y n t h e s i s a t stage 2. However, a decreased requirement f o r l y s i n e f o r h i s t o n e s y n t h e s i s i s u n l i k e l y i n stage 2 salmon t e s t e s , because the r e l a t i v e c o n c e n t r a t i o n of l y s i n e - r i c h h i s t o n e I and h i s t o n e T i n c r e a s e s d u r i n g an e q u i v a l e n t phase of t e s t i s m a t u r a t i o n i n t r o u t , w h i l e t h a t of V arginine-rich histone IV decreases (30,31). Because these histone fractions synthesized in stage 2 testes are so highly enriched with lysine, (histone I -32.1 % lysine, histone T -22.9 % lysine (20)) the need for lysine in stage 2 testes may be greater than in stage 1 testes. If this i s true, the 41 % enrichment of stage 2 testis tRNA preparations with lysine tRNA would f u l f i l l the demand for a greater a v a i l a b i l i t y of lysine in stage 2 testes for the synthesis of lysine-rich histones. The ratio of arginine tRNA to lysine tRNA in testes seems to correlate well with the ratio of protamine synthesis to histone synthesis (table 10) in a l l phases of testis develop-ment except stage 2. Although the protamine to histone ratio increased from stage 1 to stage 2, the decrease in the arginine tRNA to lysine tRNA ratio from stage 1 to stage 2 i s l i k e l y due to the enrichment of stage 2 testis tRNA populations with lysine tRNA which i s required for the lysine-rich histone synthesis of stage 2 testes discussed above. A tRNA preparation from a stage 3 testis exhibited i n -creased arginine acceptor activity (16.4 %) and significantly decreased lysine acceptor activity (53 %) when compared to a stage 2 testis tRNA preparation (table 9 and figure 13). A drastic decrease in histone synthesis was noted in stage 3 testes for only 5.4 % of total protein synthesis in stage 3 testes was histone, whereas, in stage 2 testes, 18.1 % of total protein synthesis was histone (table 2). Stage 3 testes syn-thesize large amounts of protamine and therefore require that i n ro Table 10. A comparison of the arginine and lysine acceptance acti v i t i e s of various testis tRNA preparations Basic Nuclear Protein Synthesis Protamine/Histone 1 80.9 31.9 2.53 0.02 1A 91.7 39.2 2.34 -2 93.1 41.4 2.25 0.98 3 131.7 29.1 4.53 3.04 4A 132.5 39.5 3.35 -4B 94.8 28.6 3.31 1.50 Protamine synthesis to histone synthesis ratios were taken from table 2. Arginine and lysine acceptance a c t i v i t i e s were taken from table 9. Stage Amino Acid Acceptance  Testes Arginine Lysine Arginine/ (pmoles/A26o unit) (pmoles/A26o unit) Lysine Figure 13. Relative amounts of the various tRNA's during maturation of salmon testes. large quantities of arginine are available for the synthesis of protamine. However, because histone synthesis has been drastically decreased, stage 3 testes require less lysine than stage 2 testes. Changes in the testis tRNA population correlate with these needs, because stage 3 testes have an increased content of arginine tRNA and a decreased contnet of lysine tRNA. Also, the highest arginine tRNA to lysine tRNA ratio i s found in stage 3 testes, a stage of testes which correspondingly has the highest protamine synthesis to histone synthesis ratio. Once again in stage 4 testes, the relative content of arginine and lysine tRNA seems adapted to the ratio of synthesis of the two basic nuclear proteins, histone and protamine. Protamine synthesis decreased dramatically from a value of 16.4 % of total protein synthesis in stage 3 testes to a value of 3.0 % of total protein synthesis in stage 4 testes. Histone synthesis decreased from a value of 5.4 % of total protein synthesis in stage 3 testes to a value of 2.0 % of total pro-tein synthesis in stage 4 testes. The disproportionately large decrease in protamine synthesis caused the protamine to his-tone ratio to decrease from the stage 3 value of 3.04 to a stage 4 value of 1.50. The arginine tRNA to lysine tRNA ratios of the two stage 4 testes (A & B) were decreased from the stage 3 value of 4.53 to values, 3.35 and 3.31, respectively. Thus once again the arginine tRNA to lysine tRNA ratio adapts to f i t the changes protamine synthesis to histone synthesis ratio. 138. VI. Isoaccepting forms of tRNA A r g in salmon testes at the  various stages of development As was noted above salmon testis tRNA preparations dis-played an increase in their arginine tRNA content during the period of protamine synthesis. If certain of the six arginine codons were to predominate in protamine mRNA, one might expect also to detect quantitative or qualitative differences in specific arginine tRNA's during testis maturation. For example, arginine tRNA species corresponding to the predominant arginine codons might be expected to increase disproportionately over the other arginine tRNA species during the period of rapid protamine synthesis. (a) BD-cellulose Chromatography of tRNA A r g (i) Whole Cell tRNA A r g In order to compare the amounts of individual arginine tRNA species in testes at different stages of maturation, testis tRNA was fractionated on BD-cellulose columns and the arginine acceptor profiles of these columns were compared for quantita-tive or qualitative differences. However, i f testis tRNA is eluted from BD-cellulose columns with an increasing salt gradient containing 0.01 M magnesium chloride, as suggested by Gillam et a l . (267) , considerable " t a i l i n g " of tRNA occurs in the high salt region. The elution profile of stage 4 testis tRNA chromatographed under these conditions is seen in figure 14A. When arginine acceptance assays were repeated on fractions Figure 14. Elution profiles of stage 4 testis tRNA prepara-tions on BD-cellulose in the presence and absence of dimethylformamide A. Stage 4 testis tRNA (460 A 2 6 0 units) was chromato-graphed at 23° on a column (100 x 1.2 cm) of BD-cellulose at a flow rate of 1.5 ml/min using a linear gradient formed from 1 l i t e r each of 0.45 M NaCl and 1.0 M NaCl in 0.01 M MgCl 2. Fractions were 7.8 ml. At the end of the gradient (tube 276) elution was continued with a solution of 1.0 M NaCl-0.01 M MgCl2-10 % (v/v) ethanol. B. Stage 4 testis tRNA (53 0 A 2 6 0 units) was chromato-graphed at 23° on a column (100 x 1.2 cm) of BD-cellulose at a flow rate of 1.2 ml/min using a linear gradient formed from 1 l i t e r each of 0.45 M NaCl and 1.0 M NaCl in 0.01 M MgCl2 and 2 % (v/v) dimethylformamide. Fractions were 10 ml. At the end of the gradient (tube.206) elution was continued with a solution of 1.0 M NaCl-0.01 M MgCl2-2 % (v/v) dimethylformamide-10 % (v/v) ethanol. Samples of fractions of both columns were f i r s t d i -luted with an equal volume of d i s t i l l e d water and then assayed for arginine acceptor activity using the procedure described in Materials and Methods (p. 71). Absorbance of each fraction at 260 nm (solid line); arginine acceptor activity (dotted l i n e ) . 139a 140. 1 to 169, the arginine acceptor a c t i v i t i e s obtained were similar to those detailed in figure 14A. The arginine acceptor ac t i v i t i e s of fraction 17 0 to 28 0 were not included in figure 14A because their arginine acceptance values fluctuated greatly when assayed at different times. For example, fraction #230 accepted 8 6 pmoles arginine/ml, 35 pmoles arginine/ml and 0 pmoles arginine/ml when assayed on three different occasions. However, i f 2 % (v/v) dimethylformamide was included in the eluting solutions, the "t a i l i n g " of testis tRNA on a BD-cellulose column was minimized (figure 14B). When an arginine accep-tance assay was repeated on any fraction of column B, the arginine acceptor activity obtained was similar to that detailed in figure 14B. Thus, testis tRNA fractionated on BD-cellulose in the presence of 2 % (v/v) dimethylformamide showed no anomalous results for arginine acceptance in the high salt region. It should also be noted that in the presence of 2 % (v/v) dimethylformamide, tRNA fractions are eluted from BD-cellulose columns at considerably lower salt concentrations than in i t s absence. For example, the major peak of arginine tRNA was eluted from approximately 0.60 M NaCl to 0.64 M NaCl in the presence of 2 % dimethylformamide and from approximately 0.78 M NaCl to 0.84 M NaCl in i t s absence. Also, because tRNA fractions were eluted at lower salt concentrations in the presence of 2 % dimethylformamide, the fractions eluted with the aid of 10 % ethanol from column B contained much less ultraviolet absorbing material than the fractions from column A. The arginine acceptor profile of various testis tRNA preparations chromatographed on BD-cellulose columns in the presence of 2 % dimethylformamide are compared in figure 15. To eliminate the possibility of changes in testis arginyl-tRNA synthetases during testis maturation which would affect arginine acceptor profiles, a salmon li v e r arginyl-tRNA syn-thetase preparation was used to assay the arginine acceptor activity of a l l BD-cellulose column fractions. Preliminary tests for arginine acceptor activity on samples from tubes 76 and 97 of stage 3 tRNA fractionated on a BD-cellulose column, determined the concentration of the salmon liver arginyl-tRNA synthetase and incubation time needed to assure a linear response to added tRNA. Therefore, the nanomoles of arginine accepted by each fraction of a BD-cellulose column represented the nanomoles of arginine tRNA in each fraction. No quantita-tive or qualitative difference was noted in the arginine acceptor profiles of tRNA of stage 2, stage 3, and stage 4 testes chromatographed on BD-cellulose columns. Data of table 11 indicated that 89 to 90 % of the arginine tRNA of these three stages was contained in the major peak, while only 10 to 11 % was in the minor peak. During the phase of testis maturation from stage 2 to stage 3, a proportional increase in both types of arginine tRNA is indicated because the pro-portion of the two arginine tRNA's remained the same as the arginine tRNA content increased from 60.8 to 84.3 nanomoles arginine tRNA per 500 A 2so units. A comparison of arginine acceptor profiles of stage 1, stage 1A and stage 2 indicated 142. Figure 15. A comparison of the arginine acceptor profiles of tRNA from various stages of testes chromatographed on BD-cellulose columns. Testes used to prepare each of the tRNA samples were f u l l y described in table 9. Stage 4 of this figure is the same tRNA preparation called stage 4A in table 9. The quantity of testis tRNA from each stage fractionated on a BD-cellulose column was as follows: stage 1, 340 A 2 6 0 units, stage 1A, 594 A 2 6 o units, stage 2, 517 A 2 6 0 units, stage 3, 596 A 2 6 o units, and stage 4, 53 0 A 2 6 0 units. Each testis tRNA preparation was chromatographed at 23° on a column (100 x 1.2 cm) of BD-cellulose with a linear gradient formed from 1 l i t e r each of 0.45 M NaCl and 1.0 M NaCl in 0.01 M MgCl2 and 2 % (v/v) dimethylformamide. The flow rates of the columns were as follows: stage 1, 1.3 ml/min, stage 1A, 1.1 ml/min, stage 2, 1.7 ml/min, stage 3 and stage 4, 1.2 ml/min. The size of the fractions collected were as follows: stage 1, 10.8 ml, stage 2, 9.0 ml, stage 3 and stage 4, 10 ml. The size of fractions varied in stage 1A; tubes 1 to 60 were approximately 9.0 ml, tubes 61 to 122 were approximately 8.0 ml and tubes 123 to 18 0 were approximately 11.0 ml. At the end of the gradient, elution was continued with a solution of 1.0 M NaCl - 0.01 M MgCl2 - 2 % (v/v) dimethylformamide - 10 % (v/v) ethanol. Samples of fractions were f i r s t diluted with an equal volume of d i s t i l l e d water and then assayed for arginine acceptor activity with a salmon li v e r arginyl-tRNA synthetase preparation using the pro-cedure described in Materials and Methods (p. 71). In areas with l i t t l e arginine acceptor activity, every f i f t h tube was assayed for arginine acceptance. In the areas where sizeable arginine acceptor ac-t i v i t y i s found at least every second, sometimes every tube, was assayed. Absorption of each fraction at 260 nm (solid line); arginine acceptor activity (dotted l i n e ) . 142a Stage 1 FRACTION NO. FRACTION NO. i 1 i • I ' i 1 1 • 1 • 1 • 1 >-V-T Stage 2 FRACTION NO. 142b 1 • 1 • 1 • 1 1 1 • 1 . 1 1 1 i - t r Stags 2 FRACTION N O . Stage 4 FRACTION NO. Table 11. A comparison of the sizes of the two arginine acceptor peaks of testis tRNA preparations chromatographed on BD-cellulose columns. Stage Total of Peaks a Major Peak Percent of Total Minor Peak Percent of Total 2 61 55 90 6.2 10 3 84 76 90 8.2 10 4 84 74 89 9.4 11 Total nanomoles arginine accepted by 500 A 26 0 units of tRNA chromatographed on a BD-cellulose column. Total nanomoles arginine accepted by a peak per 500 A2eo units of tRNA chromatographed on a BD-cellulose column. 144 . that qualitative differences existed between them. In stage 1A, a new peak of arginine tRNA appeared which eluted in a pos-i t i o n between the major and minor peaks of the other stages. An increase in arginine acceptor acti v i t y at this position in the arginine tRNA profile of stage 1 indicates the presence of this new arginine peak in stage 1 tRNA. The minor arginine tRNA peak seen in stage 2, stage 3 and stage 4 was no longer observed in the arginine acceptor profile of stage 1 and stage 1A. However, this minor arginine tRNA species may be present in the descending shoulder of the new arginine peak of stage 1 and stage 1A. Quantitation of the arginine peaks was d i f -f i c u l t in stage 1 and stage 1A due to their poor separation. The presence of a new arginine tRNA peak in stage 1A does not, however, necessarily indicate the presence of a new species of arginine tRNA. Often new peaks of amino acid acceptor activity have proved to be artifacts on further inves-tigation. Ribonuclease-cleaved tRNA molecules (271) and tRNAs lacking the adenosine terminus (320) are known to chromato-graph differently than native tRNA. The aggregation of tRNA (321) or a conformational change (3 22) in tRNA can cause the elution position of tRNA to be altered. Slight differences in the chromatographic conditions of the various BD-cellulose columns might cause alterations in the arginine acceptor pro-f i l e s . Thus, the question of whether this new peak represents a functionally distinct tRNA required further study. Data in table 12 indicate that the total arginine accep-tance of fractionated tRNA preparations increases from a stage 1 value of 46.8 nanomoles/500 A 2 6 0 units to stage 3 value of 84.3 nanomoles/500 A 2 6o units. This increase in the relative arginine tRNA content from stage 1 to stage 3 was similar to that observed for unfractionated tRNA samples. Because unfrac-tionated tRNA was assayed using a salmon testis arginyl-tRNA synthetase and fractionated tRNA was assayed using a salmon li v e r arginyl-tRNA synthetase the differences in the arginine acceptance values of testis tRNA preparations are independent of the source of arginyl-tRNA synthetase. The arginine accep-tance values (nanomoles arginine accepted per 1.0 A 26 0 unit) of fractionated tRNA are observed to be 15 to 30 % higher than the corresponding values for unfractionated tRNA. This d i f f e r -ence i s expected because cold TCA insoluble incorporation of arginine was used to measure the tRNA A r g content of column fractions whereas arginine incorporation into hot TCA lab i l e fraction was used to determine the tRNA A r g content of unfrac-tionated tRNA samples (see Result section p.121)„ (ii) Ribosomal-bound tRNA A r g The frequency with which different homologous species of tRNA are ribosome-bound in a c e l l may be related to the frequency of their use in protein synthesis (212,323). In stage 2 testes approximately 73 % of the arginine incorporated into proteins is used for the synthesis of protamines (table 1). Table 12. Comparison of the arginine acceptor activity of unfractionated tRNA and BD-cellulose fractionated tRNA. ci c ID C Stage Unfractionated tRNA Ratio Fractionated tRNA Ratio 1 80.9 0.61 46.8 0.56 1A 91.7 0.70 55.4 0.66 2 93.1 0.71 60.8 0.72 3 131.7 1.00 84.3 1.00 4 132.5 1.01 83.4 .99 Picomoles arginine accepted per A 2 6 o unit. Total nanomoles arginine accepted by 500 A 2 6o units of tRNA chromatographed on a BD-cellulose column. The arginine acceptance of various stage tRNAs are compared to the arginine acceptance of stage 3 tRNA. Therefore, the relative frequency with which homologous species of arginine tRNA are ribosome-bound in stage 2 testes could give a good indication of their frequency of use in protamine synthesis. Ribosomal tRNA of stage 2 testes was prepared from a ribosomal pellet containing both polysomes and monosomes by a procedure similar to the one used to isolate whole c e l l tRNA. The yield of this ribosomal tRNA from stage 2 testes was 0.073 mg/g. Because 0.307 mg/g of bulk tRNA was extracted from stage 2 testes (Table 6), ribosomal tRNA makes up approx-imately 23 % of the total tRNA in stage 2 testes. Kano-Sueoka et a l . (212) found that the polysomal and monosomal-bound tRNA of E. c o l i was approximately 30 % of a l l chargeable tRNA in the c e l l . The ribosomal tRNA fraction of stage 2 testes was found to contain 73.8 nanomoles arginine tRNA per 500 A 2 6 0 units whereas bulk tRNA of stage 2 testes contained only 60.8 nanomoles arginine tRNA per 500 A 2 6 0 units. The tRNA fraction bound to ribosomes appears 21 percent richer in arginine tRNA than the total tRNA fraction. This enrich-ment of the ribosomal-tRNA fraction with arginine tRNA species may be related to the increased frequency of occurrence of arginine in proteins synthesized by stage 2 testes. If an enrichment of ribosomal tRNA can be related to the synthesis of protamines, i.e., proteins which are 67 % arginine, this result would contradict Weil's conclusion (324) which states in yeast and E. c o l i there i s no direct relationship between the frequency of occurrence of an amino acid in proteins and the amounts of the corresponding tRNA present in the ribosomal fraction. However, further experiments are needed to solve this dispute. The ribosomal tRNA sample (Figure 16) had an arginine acceptor profile with the major peak containing 86 % of the arginine tRNA and the minor peak containing only 14 % of the arginine tRNA; a profile very similar to that of the bulk tRNA sample. This means that even though arginine tRNA species of both peaks are present more frequently on ribosomes than in the whole c e l l , the proportions at which they are present on ribosomes are similar to the proportions at which they are present in the whole c e l l . However, a l l that we can logically conclude from this study of the arginine tRNAs found bound to the ribosomes, i s that arginine tRNA species from both peaks are l i k e l y used for the synthesis of protamines. (b) RPC-5 Chromatography of tRNA A r g The separation of the arginine tRNAs of E. c o l i (325) and guinea pig l i v e r (191) by a reversed phase chromatography system (RPC-2) has revealed the presence of four isoaccepting species of arginine tRNA. Also, White (313) has noted that the profiles of charged Drosophila tRNAs separated on the recently developed reversed phase chromatography system (RPC-5) (284) gave a sharper separation of multiple isoaccepting 149. Figure 16. A comparison of the arginine acceptor p r o f i l e s of bulk tRNA and ribosomal tRNA from stage 2 testes chromatographed on BD-cellulose columns. Bulk (whole c e l l ) tRNA and r i b o s o m a l tRNA were prepared a c c o r d i n g to the procedures d e s c r i b e d i n M a t e r i a l s and Methods, pages 63 to 66 and 67, r e s p e c t i v e l y . The q u a n t i t y o f stage 2 bul k tRNA and r i b o s o m a l tRNA f r a c t i o n a t e d on a B D - c e l l u l o s e column was 517 A 2 6 0 u n i t s and 360 A 2 6 o u n i t s , r e s p e c t i v e l y . Each tRNA p r e p a r a t i o n was chromato-graphed a t 23° on a column (100 x 1.2 cm) o f BD-c e l l u l o s e w i t h a l i n e a r g r a d i e n t formed from 1 l i t e r each of 0.45 M NaCl and 1.0 M NaCl i n 0.01 M MgCl 2 and 2 % (v/v) dimethylformamide. The flow r a t e o f the columns were as f o l l o w s : bulk tRNA 1.7 ml/min and r i b o s o m a l tRNA 0.84 ml/min. F r a c -t i o n s c o l l e c t e d f o r bul k tRNA were 9.0 ml. The s i z e o f f r a c t i o n s v a r i e d f o r r i b o s o m a l tRNA. Tubes 1 to 60 were approximately 9.0 ml, tubes 61 to 135 were ap p r o x i m a t e l y 7.5 ml, and tubes 136 to 235 were ap p r o x i m a t e l y 8.5 ml. At the end of the g r a d i e n t , e l u t i o n was c o n t i n u e d w i t h a s o l u t i o n o f 1.0 M NaCl-0.01 M MgCl 2-2 % (v/v) dimethylformamide-10 % (v/v) e t h a n o l . Samples o f f r a c t i o n s were f i r s t d i l u t e d w i t h an equal volume of d i s t i l l e d water and then assayed f o r a r g i n i n e a c c e p t o r a c t i v i t y w i t h a salmon l i v e r a r g i n y l - t R N A s y n t h e t a s e p r e p a r a t i o n u s i n g the p r o -cedure d e s c r i b e d i n M a t e r i a l s and Methods (p. 71 ). Absorbance of each f r a c t i o n a t 260 nm ( s o l i d l i n e ) ; a r g i n i n e a c c e p t o r a c t i v i t y (dotted l i n e ) . 149a Stage 2 Bulk tRNA FRACTION NO. 3-0r 2-8h Stage 2 Ribosomal tRNA A0-& •JO-6 S 08 F c o 0.01 140 160 205 -to-2 10% Elhmol JO.O 225 245 species and less overlap of adjacent peaks than profiles of Drosophila tRNAs separated by BD-cellulose chromatography. Salmon testis tRNA was, therefore, separated on RPC-5 columns in the hope of better resolving the isoaccepting species of arginine tRNA. Of the gradients tried (0.50 M to 0.80 M NaCl, 0.55 M to 0.75 M NaCl, 0.60 M to 0.75 M NaCl and 0.55 M to 0.65 M NaCl), the best resolution of arginyl-tRNAs from salmon teste on RPC-5 columns was obtained using a very shallow gradient (0.55 M to 0.65 M NaCl). Using this very shallow gradient, arginyl-tRNA from stage 3 testes was fractionated on a RPC-5 column into five distinct peaks (Figure 17). The shoulder at the descending edge of peak I indicates the presence of an additional arginyl-tRNA peak. However, even shallower gradients, 0.575 M to 0.650 M NaCl and 0.575 M to 0.640 M NaCl, used for RPC-5 columns of figure 19 and figure 18, respectively, failed to resolve this shoulder into a distinct peak. The isoaccepting species of arginine tRNA were much better resolved using RPC-5 columns than BD-cellulose columns because the two peaks of arginine acceptor activity seen on BD-cellulose columns were resolved on RPC-5 columns into five possibly six, components. Qualitative differences have been observed between the arginine tRNA profiles of stage 1 and stage 3 testis tRNA on BD-cellulose columns (Figure 15). These two stages of testes have also shown a very different need for arginine in 151. Figure 17. RPC-5 p r o f i l e of stage 3 salmon t e s t i s arginyl-tRNA Transfer RNA from stage 3 testes was acylated with L-[U- 1 **C]-arginine ( s p e c i f i c a c t i v i t y , 316 mCi/mmole, Schwarz/Mann) using a salmon l i v e r aminoacyl-tRNA synthetase preparation. A t o t a l of 2.0 A 2 6o units of [ 1^C]-arginyl-tRNA (97,500 counts/ min) was fractionated on the (0.9 x 12 cm) RPC-5 column at 37°. Fractions (0.5 ml) were eluted at a rate of 12 ml/h with a 100 ml solut i o n contain-ing a l i n e a r gradient of NaCl (0.55 to 0.65 M) and other components described under Materials and Methods. The r e s i d u a l arginyl-tRNA was eluted by washing the column with a solut i o n containing 1.5 M NaCl and other components described under Materials and Methods. Samples (0.2 ml) of column f r a c t i o n s were dried on f i l t e r paper discs and counted. 6 \ <2000-z 1.5 M NaCl •65 ur D z loocH •60 •50 TOO FRACTION 150 No. 200 the synthesis of basic nuclear proteins (Table 2). Arginine was used mainly for the synthesis of histones in stage 1 testes and mainly for the synthesis of protamines in stage 3 testes. Consequently, the arginyl-tRNAs of stage 1 and stage 3 testes were cochromatographed on a RPC-5 column in the hope that the improved resolution of arginine tRNA species on RPC-5 would show i f any real difference existed between the arginine tRNA species of these stages. The arginyl-tRNA profiles of stage 1 and stage 3 testis tRNA chromatographed on a RPC-5 column are shown in figure 18. When the arginyl-tRNA profiles of these two stages of testes are compared, both arginyl-tRNA preparations exhibit a similar number of arginyl-tRNA peaks, suggesting that the extra shoulder of arginine acceptor acti v i t y seen with a stage 1 tRNA preparation of BD-cellulose i s an a r t i f a c t . Because each sample of tRNA was chromatographed separately on a BD-cellulose column, slight differences in the chromatographic condition of the BD-cellulose columns might have caused the alterations seen in the arginine acceptor profiles of stage 1 and stage 1A testis tRNA. However, stage 1 and stage 3 samples of arginyl-tRNA were fractionated simultaneously on RPC-5 columns, eliminating any variation in column chromato-graphic conditions and giving arginyl-tRNA profiles having no qualitative differences. 153. Figure 18. RPC-5 profiles of arginyl-tRNAs from stage 1 and stage 3 testes. Transfer RNA isolated from stage 1 testes and acylated with L-JU-1''C]-arginine (specific activity, 31- mCi/mmole, Schwarz/Mann) ( • ) was cochromatographed at 37° on a (0.9 x 12 cm) RPC-5 column with tRNA isolated from stage 3 testes and acylated with L-[G- 3H]-arginine (specific activity, 542 mCi/mmole, New England Nuclear) (—). Each tRNA sample was acylated with the same salmon l i v e r aminoacyl-tRNA synthetase preparation. A total of 84 ,051 counts/min stage 1 [1''C]-arginyl-tRNA and 86,343 counts/min stage 3 [3H]-arginyl-tRNA was applied to the RPC-5 column. Fractions (0.6 ml) were eluted at a rate of 12 ml/h with a 120 ml solution containing a linear gradient of NaCl (0.575 to 0.64 M) and other components described under Materials and Methods. The residual arginyl-tRNA was eluted by washing the column with a sol-ution containing 1.5 M NaCl and other components described under Materials and Methods. Samples (0.2 ml) of column fractions were pipetted into v i a l s containing 5 ml of Aquasol and counted by liquid s c i n t i l l a t i o n techniques using two channels. Values within the insert represent the ratio of [3H]-arginyl-tRNA to [ 1 4C]-arginyl-tRNA of column fractions. Although no qualitative differences are observed, quan-ti t a t i v e differences between the arginyl-tRNA profiles of stage 1 and stage 3 testis tRNA on a RPC-5 column are noted. These quantitative differences are clearly seen when ratios of [ 3H]-arginyl-tRNA (stage 3) to [ 1 **C]-arginyl-tRNA (stage 1) for each peak are examined (insert, figure 18). These differences are not artifacts due to contamination of one of the isotopic forms of arginine or artifacts due to concentration effects arising from differences in specific activity of [ 3H]- or [^C]-labeled arginine because no difference in the arginyl-tRNA profiles was found when a stage 3 testis tRNA preparation was aminoacylated with both [ 3H]-arginine and [1''C]-arginine and then cochromatographed on a RPC-5 column (Figure 19). A quantitative analysis of the arginyl-tRNA patterns of figure 18 are presented in table 13. The percentages of arginyl-tRNA in each peak were determined from the total amount of radioactivity present in the six fractions ( = 100 % ) . The percentages indicate that even though the relative concentra-tion of arginine tRNA in peaks I, II, III, and IV has increased in stage 3 tRNA preparations while the relative concentration of arginine tRNA in the ascending and descending shoulder of peak I (equivalent to peak V and VI, respectively of figure 18) has decreased in stage 3 testis tRNA preparations, the relative proportions of the six components in stage 1 and stage 3 testis tRNA preparations have not changed drastically. In fact, the largest difference between these two stages was 155. Figure 19. RPC-5 profiles of arginyl-tRNAs from stage 3 testes Transfer RNA isolated from stage 3 testes was acylated separately with L-[U-C]-arginine (specific activity, 316 mCi/mmole, Schwarz/Mann) and with L-[G- 3H]-arginine (specific activity, 542 mCi/mmole, New England Nuclear) using a salmon liv e r aminoacyl-tRNA synthetase preparation. A total of 47,534 counts/min of [ 1 C]-arginyl-tRNA and 46,668 counts/min [3H]-arginyl-tRNA were co-chroma tographed at 37° on a (0.9 x 12 cm) column. Fractions (0.6 ml) were eluted at a rate of 12 ml/h with a 100 ml solution containing a linear gradient of NaCl (0.575 to 0.65 M) and other components described under Materials and Methods. The resid-ual arginyl-tRNA was eluted by washing the column with a solution containing 1.5 M NaCl and other components described under Materials and Methods. Samples (0.2 ml) of column fractions were pipetted into v i a l s containing 5 ml of Aquasol and counted by liquid s c i n t i l l a t i o n techniques using two chan-nels. Values within the insert represent the ratio of [3H]-arginyl-tRNA to [ 1*C]-arginyl-tRNA of column fractions. in in .1000H i i < z of I < I I 1.2-1.0' 0.8' ini F R A C T I O N N o . '00 £ 6 1.5M N a C l •I 1 h-650 o CL c c D o O 50CH r"625 • 1 1 I o z A / 1 I u •600 \ V. —I— 50 100 — I — 150 •575 FRACTION No. Table 13. A comparison of the relative amounts and actual amounts of specific arginyl-tRNAs in stage 1 and stage 3 testis tRNA preparations. Peak Stage 1 Testis tRNA Stage 3 Testis tRNA Actual Amount Relative Amount _ b Actual Amount Relative Amount I 37.8 46.7 65.4 49.7 II 13.8 17.0 23.0 17.5 III 4.6 5.7 9.0 6.8 IV 3.2 3.9 5.4 4.1 V 10.3 12.8 11.6 8.8 VI 12.5 15.4 17.5 13.3 Picomoles arginyl-tRNA/A26o unit. Percentage of total arginyl-tRNA. seen with the arginyl-tRNAs of peak V; the relative amount decreased from 12.8 % in stage 1 to 8.8 % in stage 3, a d i f f e r -ence of approximately 34 %. Because approximately equal amounts of radioactive arginyl-tRNA from stage 1 testes and stage 3 testes were co-chromatographed on a RPC-5 column (3H/ll4C = 1.03), results from figure 18 indicate only the number of arginine-tRNA species present and the relative amount of each. In order to reflect accurately on the amount of each arginyl-tRNA which i s present in the testis tRNA preparation, the arginine acceptor act i v i t y in each preparation must be considered. Data of table 9 i n -dicate that there i s 1.63 times more arginine tRNA in a stage 3 testis tRNA preparation than in a stage 1 testis tRNA prep-aration. Therefore, to obtain arginyl-tRNA profiles indicat-ing the exact amount of each arginyl-tRNA species in stage 1 and stage 3 testis tRNA preparations, the [ 3H]-radioactivity value and the [ 1 l*C]-radioactivity value for each 0.2 ml sample of the RPC-5 fractions of figure 18 was multiplied by 1.63 and 1.00, respectively, and replotted (Figure 20). Also, to obtain the actual amounts of each arginyl-tRNA present in stage 1 and stage 3 testis tRNA preparations, the percentages of arginyl-tRNA in each peak (Table 13) were multiplied by the arginine tRNA content of each preparation (stage 1, 8 0.9 nmoles arginine tRNA/A2eo unit and stage 3, 131.7 nmoles arginine tRNA/A26o unit). The actual amount of each arginyl-tRNA type in stage 1 and stage 3 testis tRNA preparations i s pre-sented in table 13. 158 Figure 20. Arginyl-tRNA profiles on a RPC-5 column indicating the exact amount of specific arginyl-tRNAs in stage 1 and stage 3 testis tRNA preparations. [ 1 1 4C]-arginyl-tRNA (stage 1) i s represented by the dotted line. [3H]-arginyl-tRNA (stage 3) is represented by the dashed line. 158a 3000H X CO 2000H CN d o_ -1000 c D o 100 150 F R A C T I O N N O . From both f i g u r e 20 and t a b l e 13, one can observe t h a t a stage 3 t e s t i s tRNA p r e p a r a t i o n c o n t a i n s a g r e a t e r amount of a r g i n i n e tRNA i n a l l s i x ar g i n y l - t R N A peaks than a stage 1 t e s t i s tRNA p r e p a r a t i o n . However, whereas the amount of a r g -i n i n e tRNA i n peaks I, I I , and I I I o f a stage 3 t e s t i s tRNA p r e p a r a t i o n i n c r e a s e d * by 54 %, 50 %, and 74 %, r e s p e c t i v e l y , the amount of a r g i n i n e tRNA i n peak IV and the sh o u l d e r s o f peak I ( e q u i v a l e n t to peak V and VI) i n c r e a s e d * by o n l y 33 %, 12 % and 33 %, r e s p e c t i v e l y . T h e r e f o r e , i n a stage 3 t e s t e s tRNA p r e p a r a t i o n t h e r e seems to be a p r e f e r e n t i a l i n c r e a s e i n the amount of s p e c i f i c a r g i n y l - t R N A s making up peaks I, I I , and I I I . V I I . Codons Recognized by Salmon T e s t i s Arginyl-tRNAs (a) S y n t h e s i s , P u r i f i c a t i o n and C h a r a c t e r i z a t i o n o f Codons  by Arginyl-tRNAs Because the t r i n u c l e o t i d e s CGA, CGC, CGG, CGU, AGA, and AGG which have been i d e n t i f i e d as a r g i n i n e codons (293,326, 327) were not a v a i l a b l e commercially, these t r i n u c l e o t i d e s were s y n t h e s i z e d from the a p p r o p r i a t e d i n u c l e o s i d e monophos-phates and n u c l e o s i d e 5'-diphosphates u s i n g primer-dependent p o l y n u c l e o t i d e phosphorylase from M i c r o c o c c u s l y s o d e i k t i c u s * I n c r e a s e s were c a l c u l a t e d by t a k i n g the d i f f e r e n c e between stage 1 and stage 3 v a l u e s and d e t e r m i n i n g i t s percentage of the average between the two v a l u e s . The reaction used, can be described as follows: XPY + NDP polynucleotide, p . ( 1 ) y phosphorylase ^ r In equation (1), XpY i s a dinucleoside monophosphate, NDP, a nucleoside 51-diphosphate, and XpYpN, the trinucleoside diphosphate product. The commercially available dinucleoside monophosphates, ApG and CpG, were used for trinucleotide syn-thesis without further purification because at least 97 per-cent of each dinucleoside monophosphate migrated as one spot in two paper chromatography systems, A and B. The identity of these dinucleoside monophosphates was confirmed by the molar base ratios of their RNase T 2 and snake venom phosphodiesterase digestion products. Maximal synthesis of the trinucleotides was obtained by fixing the ratio of dinucleoside monophosphate to nucleoside 51-diphosphate at 6 to 1 (282) and by including the appropriate concentration of sodium chloride (0.4 M) in the reaction mixture (328). The products were not separated from the reactants by paper chromatography, as i s the usual procedure, but rather by DEAE-cellulose chromatography using a linearly increasing concentration of N I U H C O 3 (pH 8.0) (284). Purification of the trinucleotide CpGpG by DEAE-cellulose chrom-atography i s illustrated in figure 21. The trinucleotide, CpGpG, is well resolved from both reactants, CpG and GDP. Other possible reaction products such as the tetramer, CpGpGpG, and poly G are separated from the trinucleotide, CpGpG, being eluted from the column with 1 M NH\HC03. The yield of t r i -nucleotides (calculated as the percentage mononucleotide 161. Figure 21. Purification of the trinucleotide CpGpG by DEAE-cellulose chromatography. The reaction mixture containing CpGpG (2 ml) (described in Materials and Methods) was diluted with 38 ml of water and applied to a column (1.2 x 50 cm) of DEAE-cellulose (bicarbonate form). Nucleotides were eluted from the column at a flow rate of 1.4 ml/min with a linear gradient formed from 1 l i t e r each of d i s t i l l e d water and 0.175 M N I U H C O 3 (pH 8.0). Residual nucleotides remaining on the column were eluted with a solution of 1 M NR\HC03 (pH 8.0). Fractions (8.2 ml) were monitored at 260 nm. recovered as trinucleotide) varied from the minimum value of 30.2 % for ApGpG synthesis to a maximum value of 72.4 % for CpGpU synthe s i s . The chromatographic mobilities of synthesized trinucleo-side diphosphates are tabulated in table 14. A l l trinucleo-tides except the two marked with asterisks migrated as single ultraviolet-absorbing spots in both chromatogrpahic systems. The Rf 3'-UMP values of contaminants of the two trinucleotides marked with asterisks, as well as estimates of the percent contamination are given in table 15. Data from table 15 i n -dicate that the trinucleotide, CpGpU, i s contaminated 2.4 % as shown by chromatography in system D. Data from table 15 also indicate that CpGpG i s contaminated 29.6 % as shown by chromatography in system C and 18.9 % as shown by chromatography in system D. However, the contaminants of CpGpG in both chromatography systems remain at the origin and when eluted give spectra identical to CpGpG at pH 1.0, pH 7.0, and pH 11.0. The ultraviolet-absorbing material at the origin i s l i k e l y not a true contaminant but rather an aggregate of CpGpG formed through the C-G and G-G base interaction of many trinucleotide molecules. This aggregation phenomenon of CpGpG may also explain the apparent difference in percent contamination of CpGpG in the two chromatographic systems. Because aggre-gates of CpGpG seem to be the only contaminant of the CpGpG preparation and because a l l other trinucleotide preparations are at least 97 percent pure, a l l six trinucleotides are suit-Table 1 4. Summary of the Rf values of the trinucleotides Compound Rf 5'-AMPa Solvent A B C D ApGpA 0 . 7 8 ( 0 . 8 1 ) d 0 . 1 1 ( 0 . 1 5 ) d - - - -ApGpG 0 . 5 4 - 0 . 1 1 - - - - -CpGpA 0 . 7 6 ( 0 . 8 1 ) e 0 . 4 7 ( 0 . 4 7 ) e . - - - -CpGpC - - - - 0 . 5 3 ( 0 . 4 2 ) f 0 . 8 2 ( 0 . 9 3 ) f CpGpG* - - - 0 . 4 7 ( 0 . 4 4 ) f 0 . 4 8 ( 0 . 5 6 ) f CpGpU* - - - - 0 . 6 5 ( 0 . 5 7 ) f 0 . 5 4 ( 0 . 6 3 ) f Rf 3'-UMPb Solvent 0 a The Rf 5'-AMP i s the mobility relative to that of 5'-AMP. b The Rf 3'-UMP i s the mobility relative to that of 3'-UMP. The composition of solvents is given in Materials and Methods. Rf 5'-AMP of trinucleotide reported in reference 2 8 3 . Rf 5'-AMP of trinucleotide reported in reference 2 9 2 . ^ Rf 3'-UMP of trinucleotide reported in reference 3 2 9 . Except for those compounds marked with an asterisk, 2 . 0 A 2 6 0 units of each t r i -nucleotide migrated as a single spot in both solvents. The contamination of the com-pounds marked with asterisks i s described further in text and table 1 5 . 164. Table 15. Contamination of t r i n u c l e o t i d e s Compound Trinucleotide Rf 3'-UMPa Contaminant Rf 3*-UMPa Percent Contamination*1 Solvent C D Solvent C D Solvent C D CpGpG 0.47 0.48 o r i g i n o r i g i n 29.6 18.6 CpGpU 0.65 0.54 0.76 2.4 See footnote b, Table 14. See section on Materials and Methods. Percent contamination i s defined as follows: x absorbance of eluates of the contaminant absorbance of eluates of t r i n u c l e o t i d e and contaminant able for use in ribosome binding assays without further pur-i f i c a t i o n . Base ratio determination and sequence analysis of T2-RNase and venom phosphodiesterase digestion products (Tables 16 and 17) confirmed the identity of each trinucleotide. In both tables the deviations of the experimentally determined product ratio from the theoretical values are compatible with the expected experimental error. (b) Codon Recognition by Unfractionated Arginyl-tRNA It is known that while the codon assignments do not change from organism to organism, the extent to which the i n -dividual codons in each set i s actually used may di f f e r mark-edly (297). A comparison of the binding responses of an aminoacyl-tRNA to ribosomes in the presence of trinucleotides of a codon set indicates the actual use of individual codons of the set by an organism. Consequently, i t was thought that the response of salmon testis arginyl-tRNA in trinucleotide-stimulated binding reactions would give some indication of the actual use of each arginine codon in a salmon testis c e l l . An arginyl-tRNA preparation obtained by charging unfrac-tionated stage 3 testis tRNA was tested for binding in the presence of trinucleotides CGX ( X = A, C, G, or U) and AGY ( Y = A or G). The responses of arginyl-tRNA from salmon testes to these trinucleotides are shown in table 18. For comparative purposes, responses of guinea pig l i v e r and E. c o l i arginyl-tRNA preparations to these trinucleotides (330) are VD VD Table 16. Characterization of t r i n u c l e o t i d e s by RNase T 2 digestion and estimation of molar r a t i o s Compound A.U. degraded Chr. system A Ap C CP G Gp U Theoretical molar r a t i o s 1 3 ApGpA 2.5 C 0.88 1.00 - - 1.03 1:1:1 ApGpG 2.2 H 1.00 - 1.04 0.89 1:1:1 CpGpA 2.5 H 1.10 1.00 - 0.78 1:1:1 CpGpC 2.7 E 1.14 0.96 - 1.00 1:1:1 CpGpG 2.2 F - - - 0.92 1.00 1.09 1:1:1 CpGpU 3.1 F - - - 0.97 - 1.10 1.00 1:1:1 The chromatography systems used to separate the digestion products are described i n Materials and Methods. Theoretical molar r a t i o s of the nucleosides and nucleotides reading i n order from l e f t to r i g h t across the table. r-Table 17. Characterization of t r i n u c l e o t i d e s by venom phosphodiesterase digestion and e s t i -mation of molar r a t i o s Compound A.U. degraded Chr. system A pA C pC G pG pU Theoretical molar ratios* 3 ApGpA 2.5 I. 1.05 1.00 - 0.89 1:1:1 ApGpG 2.2 G 1.00 - - 1.89 1:2 CpGpA 2.5 H 0.88 1.10 - 1.00 1:1:1 CpGpC 2.7 E 0.95 1.14 - 1.00 1:1:1 CpGpG 2.2 C 1.00 - 2.17 — 1:2 CpGpU 3.1 C 0.97 - 1.10 1.00 1:1:1 The chromatography systems used to separate the digestion products are described in Materials and Methods. Theoretical molar ratios of the nucleosides and nucleotides reading in order from l e f t to right across the table. Table 18. Activity of unfractionated arginyl-tRNA from stage 3 salmon testes in trinucleotide-stimulated binding to E. c o l i ribosomes Trinucleotide A pmoles [1*C]-arginyl-tRNA bound to ribosomes3 Salmon testis tRNA b c (stage 3) Guinea pig liver tRNA E. c o l i tRNA° ApGpA 0.04 0.12 0.10 ApGpG 0.83 0.63 0.12 CpGpA 1.98 1.28 1.09 CpGpC 1.29 0.67 0.47 CpGpG 0.37 0.97 0.20 CpGpU 1.99 0.81 0.90 None (0.98) (1.23) (1.27) The change in the number of picomoles, A pmoles, was obtained by subtracting [1I*C]-arginyl-tRNA bound to ribosomes without trinucleotides from that bound with t r i -nucleotides. The number of pmoles of [1''C]-arginyl-tRNA bound to ribosomes in the absence of trinucleotides i s enclosed within parentheses. These reactions contained the components described under Materials and Methods, and 14.5 pmoles of [1"c]-arginyl-tRNA. For comparative purposes, previous results with guinea pig liver and E. c o l i arginyl-tRNA are shown also (33 0). Reactions for salmon testis and guinea pig liver arginyl-tRNA contained 0.02 M Mg + + and reactions for E. c o l i arginyl-tRNA contained 0.03 M Mg + +. also included in table 13. Like the arginyl-tRNA of guinea pig l i v e r and E. c o l i , arginyl-tRNA of salmon testes responded well to the codons, CGA, CGC, and CGU. Also, like the arginyl tRNA of these other two organisms, i t showed l i t t l e response to the arginine codon> AGA. The relative response of salmon testis arginyl-tRNA to AGG was high compared to that of E. c o l i arginyl-tRNA but somewhat similar to the response of guinea pig l i v e r arginyl-tRNA to AGG. The response of salmon testis arginyl-tRNA to CGG was low compared to guinea pig li v e r arginyl-tRNA but somewhat similar to the response of E. c o l i arginyl-tRNA to CGG. From the responses of salmon testis arginyl-tRNA (stage 3) in the trinucleotide-stimulated ribosome binding reactions, one can predict that the major portion of arginyl-tRNA from salmon testes consists of arginyl tRNA species corresponding to the codons CGA, CGC, CGU, and AGG, One can also predict that a small portion of arginyl-tRNA from salmon testes consists of arginyl-tRNA correspond-ing to the codon CGG and that l i t t l e or no arginyl-tRNA from salmon testes corresponds to AGA. (c) Codon Recognition by an Arginyl-tRNA Fraction from  a BD-cellulose Column As shown in figure 15, the separation of stage 3 salmon testis tRNA by BD-cellulose chromatography revealed two peaks (a major and minor) of arginine acceptor activity. Transfer RNA of tube #78 of the major arginine acceptor fraction was aminoacylated w i t h [ 1 l*C] - a r g i n i n e and the b i n d i n g of t h i s [ 1 **C] - a r g i n y l - t R N A to E. c o l i ribosomes assayed i n the presence of the s i x a r g i n i n e codons. The r e s u l t s of these assays are g i v e n i n t a b l e 19. A r g i n y l - t R N A of tube #78 responded w e l l to a r g i n i n e codons, CGA, CGC, CGU, and AGG and o n l y s l i g h t l y to CGG. The r e s u l t s o f s t u d i e s (298-331,332) on the s p e c i f i c i t y of tRNA f o r r e c o g n i t i o n of codons have p r e v i o u s l y shown t h a t w h i l e t h e r e i s a s t r i c t s p e c i f i c i t y of tRNA f o r the f i r s t two l e t t e r s of a codon, one tRNA can r e c o g n i z e m u l t i p l e codons d i f f e r i n g i n the t h i r d l e t t e r . Thus, because of the s t r i c t s p e c i f i c i t y of tRNA f o r the f i r s t l e t t e r of a codon, one must conclude t h a t the major peak of a r g i n i n e a c c e p t o r a c t i v i t y , seen on B D - c e l l u l o s e columns, c o n t a i n s a t l e a s t two a r g i n y l -tRNA s p e c i e s . These s p e c i e s of a r g i n y l - t R N A may, however, be separated from each o t h e r on RPC-5 columns because RPC-5 columns r e s o l v e a r g i n y l - t R N A i n t o many more components than do B D - c e l l u l o s e columns. (d) Codon R e c o g n i t i o n by Arginyl-tRNA F r a c t i o n s from a  RPC-5 Column I t was o f i n t e r e s t t o t e s t the s p e c i f i c i t y of c e r t a i n RPC-5 a r g i n y l - t R N A components i n o r d e r to observe i f they r e c o g n i z e d d i s t i n c t codons. Uncharged salmon t e s t i s tRNA (473 A 2 6 0 u n i t s ) was chromatographed on a RPC-5 column (2 x 50 cm) u s i n g a 2 l i t e r l i n e a r g r a d i e n t of NaCl (0.57 5 M t o 0.64 0 M) i n o r d e r to o b t a i n enough a r g i n i n e tRNA separated Table 19. A c t i v i t y of arginyl-tRNA (tube 78 of BD-cellulose column f r a c t i o n a t i n g stage 3 t e s t i s tRNA) i n t r i -nucleotide-stimulated binding to E. c o l i ribosomes. Trinucleotide A pmoles [ 1 "*C]-arginyl-tRNA bound to ribosomes c ApGpA -0.11 ApGpG 0.81 CpGpA 1.39 CpGpC 1.22 CpGpG 0.26 CpGpU 2.51 None (1.13) See footnote, table 18. Reaction contained the components described under Materials and Methods, and 15.8 pmoles of [ 1^C]-arginyl-tRNA (tube 78 of BD-cellulose column f r a c t i o n a t i n g stage 3 t e s t i s tRNA, figure 15). into i t s multiple components to charge with [ 1^C]-arginine and to test i t in trinucleotide-stimulated ribosomal binding reactions. However, when fractions from this column were assayed for arginine acceptor activity, no resolution of the multiple arginyl-tRNA components was observed. In an attempt to obtain better resolution of the uncharged arginine tRNA components, fractions of the previous column containing arg-inine acceptor act i v i t y were pooled and rechromatographed on a long (0.9 x 58 cm) RPC-5 column. The arginine acceptance profile of fractions from this RPC-5 column i s shown in figure 22. This RPC-5 column did not fractionate the part-i a l l y purified, uncharged arginine tRNA into multiple tRNA components. Likely, both these RPC-5 columns were overloaded with arginine tRNA. Also, because the separation of unamino-acylated tRNA with RPC-2 has been shown to be poorer than i t i s with aminoacylated samples (192), the separation of un-charged arginine tRNA may also be poorer on RPC-5 than i t i s with charged arginine tRNA. However, i t was reasoned that because 8 A 2 6 o units of unf ractionated [ 1 ''C] -arginyl-tRNA were resolved into multiple arginyl-tRNA components by chromatography on a (0.9 x 12 cm) RPC-5 column (figure 19), 4 0 A 2 6 0 units of unf ractionated [11*C]-arginyl-tRNA should be resolved on a (0.9 x 58 cm) RPC-5 column into various peaks of arginyl-tRNA, most of which would contain enough arginyl-tRNA to be used for ribosomal binding assays. Accordingly, 173. Figure 22. Uncharged salmon testis tRNA chromatographed on a RPC-5 column Fractions from a RPC-5 column (2 x 50 cm) (fractionating 47 3 A 2 6 0 units of salmon testis tRNA, see Result section) containing arginine acceptor activity were pooled and rechromatographed at 37° on this (0.9 x 58 cm) RPC-5 column. Frac-tions (4 ml) were eluted every 1.8 min from this column with a 800 ml solution containing a linear gradient of NaCl (0.575 M to 0.650 M) and other components described under Materials and Methods. The residual tRNA was eluted by washing the column with a solution containing 1.5 M NaCl and other components described under Materials and Methods. Samples of fractions (0.1 ml) were assayed for arginine acceptor act i v i t y with a salmon li v e r aminoacyl-tRNA synthetase preparation using the procedure described in Materials and Methods. Mixes contained 2 nanomoles arginine (0.2 yCi [^C]-arginine) and were incubated for 4 0 min. Absorbance of each fraction at 260 nm (solid line); counts/min [ 1 "*C]-arginine accepted per 0.1 ml (dotted l i n e ) . 4 0 A 2 6 O units of stage 3 testis tRNA aminoacylated with [^C]-arginine and chromatographed on a (0.9 x 58 cm) RPC-5 column were found to be resolved into four multiple arginyl-tRNA components I, II, III, and IV (figure 23). Like other RPC-5 arginyl-tRNA profiles presented in this thesis, the presence of shoulders at the ascending and descending edge of peak I (figure 23) indicate the presence of two additional arginyl-tRNA components. Three arginyl-tRNAs (I, II, and III) fractionated by the RPC-5 column (figure 23) were tested for stimulation of their binding to ribosomes in the presence of the six arginine codons. The results of the binding assays are given in table 20. Quantitative responses with fractions I and II of RPC-5 column were considerably greater than unfractionated arginyl-tRNA (Table 18) or the arginyl-tRNA fraction purified by BD-cellulose chromatography (Table 19) . The codon CGU, for example, directs stable binding to ribosomes of approximately 67 percent of the available [1''C]-arginyl-tRNA I. However, CGU directs the binding of only 14 percent and 16 percent of the available unf ractionated [ 1 "*C]-arginyl-tRNA and BD-cellulose [ 1^C]-arginyl-tRNA, respectively. However, the purity of the arginyl-tRNA fractions was not the only factor which determined the ribosome binding response. If the amount of unfractionated tRNA added to the ribosome binding assays is measured by [ 1 "*C]-arginyl-tRNA precipitated on f i l t e r paper discs by TCA rather than by [ 1^C]-arginyl-tRNA dried 175. Figure 23. A large scale RPC-5 chromatographic separation of arginyl-tRNAs from stage 3 salmon testes. Transfer RNA from stage 3 testes was acylated with L-[U- 1"c]-arginine (specific activity, 316 mCi/mmole, Schwarz/Mann) using a salmon li v e r aminoacyl-tRNA synthetase preparation. A total of 40 A 2 6o units of [ 1"c]-arginyl-tRNA (2,025,200 counts/min) was fractionated on a (0.9 x 58 cm) RPC-5 column at 37°. Fractions of (4 ml) were eluted every 2.8 min with a 8 00 ml solution con-taining a linear gradient of NaCl (0.575 M to 0.650 M) and other components described under Materials and Methods. Samples (0.1 ml) of column fractions were dried on f i l t e r paper and counted. 175a I 4000H O a. < 3000-C O) < I 1—1 O 2000H Z i z o o IOOOH 12 .... A 5 0 FRACTION No. 1 0 0 2 \ * .623 O a Z • 600 ;575 150 Table 20. Specificity of arginyl-tRNAs purified by RPC-5 chromatography in trinucleotide-stimulated bind-ing to E. c o l i ribosomes. Trinucleotide Apmoles [ 1^C]-arginyl-tRNA bound to ribosomes' II III ApGpA ApGpG CpGpA CpGpC CpGpG CpGpU None 0.29 |4.51| (0.91) 5.42 0.26 -0.04 2.15| -0.04 (0.72) 0.03 •0.03 i T ! 0.19i I j 0.02 (0.37) See footnote, table 18. Arginyl-tRNA of tube # 44 (figure 23) was designated arginyl-tRNA I; arginyl-tRNAs of tubes # 56 to 60 and tubes # 67 to 7 6 were designated arginyl-tRNA II and III, respec-tively. The fractions were concentrated by method 2 (see Materials and Methods). Reaction mixture contained the com-ponents described under Materials and Methods and either 6.78 pmoles [ 1"C]-arginyl-tRNA I, 8.16 pmoles [ 1^C]-arginyl-tRNA II or 6.75 pmoles of [ 1*C]-arginyl-tRNA III. on f i l t e r paper discs, a forty-three percent lower value i s obtained. It seems that pronounced.deacylation of unfraction-ated [1''C]-arginyl-tRNA and BD-cellulose [ 1 *C] -arginyl-tRNA has occurred during their purification and concentration; probably during lyophilization (see method 1, Materials and Methods, p. 76). Because [ 1 l*C]-arginyl-tRNA fractions from the RPC-5 column were concentrated for ribosome binding by minature DEAE-cellulose columns and ethanol precipitation rather than lyophilization, v i r t u a l l y no [ 1 UC]-arginine was hydrolyzed during concentration procedures, leaving a l l the [^C]-arginyl-tRNA aminoacylated. From results (Table 20) one can observe that over 50 percent of arginyl-tRNA I binds to ribosomes in the presence of the trinucleotides CGA, CGC, and CGU. Likely, a small amount of arginyl-tRNA (specific for CGG) contaminates this arginyl-tRNA I fraction because 5 percent of the arginyl-tRNA also binds to ribosomes in the presence of CGG. Therefore, the arginyl-tRNA I, making up approximately 50 percent of the arginyl-tRNA in stage 3 testes (Table 13) corresponds to codons CGZ ( Z = A, C, and U). For the multiple recognition-pattern observed (CGA, CGC, and CGU), the Wobble hypothesis (333,334) predicts that inosine w i l l be located in the f i r s t position of the anticodon of arginyl-tRNA I. [ 1 **C]-arginyl-tRNA II was bound in the presence of both AGG and CGG. However, the stimulation of binding of CGG (26 percent of arginyl-tRNA II bound to ribosomes) was con-siderably less than that of AGG (66 percent of arginyl-tRNA II bound to ribosomes). Thus, the arginyl-tRNA II, a fraction containing 17.5 percent of the arginyl-tRNA of stage 3 salmon testes (Table 13) responds primarily to the arginine codon AGG. According to the Wobble hypothesis (333,334) an arginyl-tRNA specifically recognizing the codon AGG would have an anticodon sequence, CpCpU. The lower response of the arginyl-tRNA II fraction to CGG may be an a r t i f a c t of the binding reaction or may indicate the existence in peak II of an additional arginyl-tRNA (specific for CGG). Arginyl-tRNA (specific for CGG) may be contained in the descending shoulder of peak I, which overlaps fractions of peak II. Further work is necessary to elucidate the origin of this CGG response of arginyl-tRNA II. [ 1 **C]-arginyl-tRNA III was bound in the presence of AGG, AGA, and to a lesser extent in the presence of CGG. However, quantitative responses with arginyl-tRNA from fractions I and II (RPC-5 column, figure 23) were considerably greater (up to 7 times greater) than with the arginyl-tRNA of fraction III. The lower responses of arginyl-tRNA III to the arginine codons could have been due to a rapid deacylation of this fraction during the incubation reaction. For this reason, the amount of [ 1 **C]-arginyl-tRNA III (counts/min in fraction precipitated by TCA) was measured before and after incubation. However, because i t was found that 68 percent of [^C]-arginyl-tRNA III remained acylated at the conclusion of the ribosome binding assay, the lower responses of arginyl-tRNA III to arginine codons are not due to rapid deacylation during incubation. Arginyl-tRNA III may be an unmodified form of argin tRNA because unmodified tRNAs sometime show a reduced a b i l i t y to bind to ribosomes (204,209). However, because of i t s late eluting position on RPC-5, arginyl-tRNA III more l i k e l y represents either an aggregated form (probably a dimer) (321) or an inactive conformational monomer (322) of another arginyl-tRNA. Aggregated tRNAs, yeast glycyl-tRNA IV and E. c o l i seryl-tRNA IV, showed l i t t l e stimulation of binding under normal conditions (332). Whereas native E. c o l i tryptophanyl-tRNA shows a response to poly (UG), the inactive conformational monomer of E. c o l i tryptophanyl-tRNA shows no response to poly (UG) (335) . A stimulation of the binding reaction was observed with these tRNAs only after they were heated to 50° or 60° for 5 min in the presence of Mg + + (to convert dimers or inactive monomers to active monomers) prior to the binding reaction. Thus, i f arginyl-tRNA III is either a dimer or an inactive monomer, heating arginyl-tRNA III to 60° for 5 min in the presence of Mg prior to the binding reaction should increase the amount bound to ribosomes in the presence of arginine codons. The position of elution of arginyl-tRNA III from a Sephadex G-100 column should indicate i t s form (dimer or inactive monomer). For instance, i f arginyl-tRNA III i s a dimer, on Sephadex G-100 chromatography i t should be eluted much earlier than the other arginyl-tRNAs of salmon testes. Further work i s also necessary to elucidate the origin of the active monomer (s) of arginyl-tRNA III. In summary, ribosome binding assays with RPC-5 arginyl-tRNA fractions have shown that arginyl-tRNA I is specific for codons CGA, CGC, and CGU; that arginyl-tRNA II is specific to codon AGG and l i k e l y contaminated with an arginyl-tRNA specific for CGG; and that arginyl-tRNA III i s l i k e l y either a dimer or an inactive monomer. Further work i s needed to elucidate the trinucleotide stimulation of binding for arginyl-tRNAs, IV, V, and VI. CONCLUSIONS Transfer RNA has been isolated from salmon testes at four distinct stages of sexual maturation. Determination of the amounts of tRNA (mg/g) in testes at these four stages of development has shown that during sexual maturation the testis tRNA population decreases proportionally to that of total testis RNA. The fact that tRNA molecules comprise approximately 10 percent of the total testis RNA throughout development i n -dicates that the RNA decrease occurring during 0. tschawytscha testis maturation must be a specific and well regulated process. Evidence i s presented in this thesis to suggest that the tRNA population of testis tissue i s specialized for the syn-thesis of basic nuclear proteins. The relative amount of arginine and lysine tRNA extracted from salmon testes i s found to be significantly greater than tRNA prepared from salmon l i v e r . This enrichment of testis tRNA populations with arg-inine and lysine accepting species seems related to the dis-proportionate requirement of arginine and lysine for basic nuclear protein synthesis in testes, a tissue with a very high DNA content. Evidence i s also presented to suggest that the lysine and arginine tRNA content of testis tissue at a specific stage of development is adapted to the type of basic nuclear protein synthesis occurring at that phase of maturation. The arginine tRNA content of testis tissue i s found to i n -crease continuously during testis maturation up to a value, at stage 3, 4 6% greater than stage 1. During this period of testis development, synthesis of basic nuclear protein i s transformed from the synthesis of mainly histone (stage 1) to the synthesis of mainly protamine (stage 3). The lysine tRNA content of testis tissue i s found to increase by 41 % during the i n i t i a l phase of testis maturation (stage 1 to stage 2), a period in which i t is believed a large portion of the histones synthesized are lysine-rich types (histone I and histone T). The lysine tRNA content of testis tissue i s then found to decrease by 53 % from stage 2 to stage 3, a phase during which the proportion of histone synthesis to protamine synthesis decreased from a 1:1 value to a 1:3 value. These differences in the relative amount of arginine and lysine tRNA appear significant because they are much larger than differences relating to biological variation between testes of a similar stage. Thus, the changes in the content of arginine and lysine tRNA observed during testis maturation suggest specialization of the testis tRNA population for the synthesis of histones and protamines. These results, together with results of others (226-23 0,232-238,240-245) suggest that the levels of the various tRNAs in cel l s are altered in a manner consistent with the u t i l i z a t i o n of the various amino acids in protein synthesis. In order to identify and quantitate the various iso-accepting forms of arginine tRNA in salmon testes at the various stages of testis development, testis tRNA preparations were chromatographed on BD-cellulose and RPC-5 columns. It was found that BD-cellulose columns fractionated arignine tRNA from salmon testes into only 2 peaks, whereas, RPC-5 columns fraction i t into 4 peaks (plus two additional shoul-ders) . Cochromatography of stage 1 and stage 3 arginyl-tRNAs on RPC-5 revealed quantitative but no qualitative d i f -ferences between the arginyl-tRNAs of these two stages. Although a l l the RPC-5 arginyl-tRNA peaks were increased in stage 3 testes, the amounts of specific arginyl-tRNA making up peaks I, II and III were preferentially increased 54 %, 50 % and 74 %, respectively. Arginyl-tRNA^., an isoacceptor comprising 50 percent of the arginyl-tRNA of stage 3 testes, responded specifically to the codons CGA, CGC, and CGU in ribosome binding assays. Arginyl-tRNAjj, comprising approx-imately 17.5 % of the arginyl-tRNA in stage 3, responded most strongly to the codon AGG in ribosome binding assays. Arginyl-tRNAj , comprising approximately 7 % of the arginyl-tRNA of stage 3 testes, i s l i k e l y either a dimer or an inactive monomer of arginyl-tRNA(s) responding to the codons AGA and AGG. This evidence suggests that arginyl-tRNA^. specific for codons CGA(C,U) and arginyl-tRNA^ specific for codon AGG preferentially increase during the phase of salmon testis maturation in which synthesis of basic nuclear proteins i s transformed from mainly histones to mainly protamines. The amounts of arginyl-tRNAj.^ in the various stages of testis is not l i k e l y related to basic nuclear protein synthesis but rather to the storage conditions of the various tRNA samples. Also, during this research, certain physical and kinetic properties of the salmon l i v e r arginyl-tRNA synthetase were determined. Experimental evidence indicated that the arginyl-tRNA synthetase isolated from salmon liver aminoacylated salmon testis tRNA with arginine in 15 min to the greatest extent at 23° and was heat labile at 37°. The formation of arginyl-tRNA by salmon li v e r arginyl-tRNA synthetase occurred to the greatest extent at NaCl concentrations between 0.125 M and 0.25 M and was inhibited 18 % in the presence of 4.8 % ethanol. After prolonged storage at -20°, salmon li v e r arginyl-tRNA synthetase preparations in 40 % glycerol retained sufficient enzyme activity to f u l l y charge the tRNA A r g present in the incubation mixture. The Km values of the salmon li v e r arginyl-tRNA synthetase for arginine and salmon testis tRNA were found to be 0.19 yM and 0.76 yM, respectively. A number of questions have been l e f t unanswered at the conclusion of this research investigating changes in tRNA d u r i n g salmon t e s t i s development. F i r s t , one may ask i f the i n c r e a s e i n a s p a r t i c a c i d and s e r i n e tRNA observed d u r i n g the i n i t i a l phase of t e s t i s m a t u r a t i o n (stage 1 t o stage 2) i s r e l a t e d to the s y n t h e s i s o f as y e t , u n c h a r a c t e r i z e d t e s t i s p r o t e i n s abundant i n these amino a c i d s . Secondly, one may ask i f the types or q u a n t i t y of l y s i n e i s o a c c e p t o r tRNAs a l t e r d u r i n g salmon t e s t i s development. T h i r d l y , one may ask i f the a c t i v i t y o f l y s y l - o r a r g i n y l - t R N A synthetase a l t e r s s i m u l t a n -e o u s l y w i t h changes i n the l y s i n e and a r g i n i n e tRNA c o n t e n t of t e s t i s t i s s u e . Hormonally induced t r o u t t e s t e s have been r e c e n t l y shown (306) to c o n t a i n more germ c e l l s a t the same stage of m a t u r a t i o n than n a t u r a l l y maturing t r o u t t e s t e s . Thus, the hormonally induced t r o u t t e s t i s system i s l i k e l y a b e t t e r system f o r f u r t h e r i n v e s t i g a t i o n o f these problems because s p e c i f i c s p e c i a l i z e d p r o t e i n s l i k e l y form a g r e a t e r p o r t i o n of t o t a l t e s t i s p r o t e i n s y n t h e s i s a t the stage of maximum s y n t h e s i s . Furthermore, a l t h o u g h t h i s and o t h e r s t u d i e s i n d i c a t e t h a t t h e r e i s a c o r r e l a t i o n between the l e v e l s of v a r i o u s tRNAs i n a c e l l and the u t i l i z a t i o n o f the v a r i o u s amino a c i d s i n p r o t e i n s y n t h e s i s , the mechansm(s) which c o n t r o l s l e v e l s o f v a r i o u s tRNAs i n c e l l s i s (are) as y e t unknown. The l e v e l s o f v a r i o u s tRNAs i n c e l l s may be a l t e r e d through s e l e c t i v e s y n t h e s i s , p r o c e s s i n g or d e g r a d a t i o n of the tRNA m o l e c u l e s . 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